# Patent

## Title
Trypanosome microsome system and uses thereof

## Abstract

The present invention provides cell free preparations of protozoan microsomes, wherein the cell free preparation of protozoan microsomes demonstrate the ability to translocate a protozoan polypeptide into the microsome, methods of preparing cell free preparations of protozoan microsomes, and methods of using cell free preparations of protozoan microsomes.

## Background
CONTINUING APPLICATION DATA
This application is a continuation-in-part of International Application No. PCT/US2007/017555, filed Aug. 7, 2007, which claims the benefit of U.S. Provisional Application Ser. No. 60/836,045, filed Aug. 7, 2006; further this application claims the benefit of U.S. Provisional Application Ser. No. 61/094,457, filed Sep. 5, 2008; all of which are incorporated herein by reference in their entireties.


GOVERNMENT FUNDING
The present invention was made with government support under Grant No. AI053086, awarded by the National Institutes of Health. The Government has certain rights in this invention.


BACKGROUND
Human African trypanosomiasis (HAT) occurs in 36 countries in Sub-Saharan Africa, threatening an estimated 60 million people with debilitating disease. No vaccines are available for prevention of infection by Trypanosoma brucei, which causes trypanosomiasis. Without chemotherapeutic treatment, T. brucei kills infected humans. Currently, there are only four drugs approved for the treatment of HAT. All are quite toxic and cause serious side effects and in some cases death (reviewed by Fairlamb, 2003, Trends Parasitol; 19(11):488-94; and Barrett et al., 2007, Br J Pharmacol; 152(8):1155-71). And drug resistance is of concern (for review, see Docampo and Moreno, 2003, Parasitol Res; 90 Supp 1:S10-3). Consequently, there is a strong need for better chemotherapeutic options for the treatment of trypanosomiasis. In addition, additional new drugs must be developed, in order to prepare for possible emergence of drug resistance in the parasites (de Koning, 2001, Int J Parasitol; 31(5-6):512-22; Ouellette, 2001, Trop Med Int Health; 6(11):874-82; and Sinyangwe et al., 2004, Vet Parasitol; 119(2-3):12-35).
Unique to T. brucei is the expression of a variable surface glycoprotein (VSG) coat on the cell surface, which undergoes constant variation in order to evade the humoral immune system and host antibodies. It is thought that recombination from a repertoire of greater that 1,000 VSG genes is responsible for the vast diversity of the parasite, and its effectiveness in immune evasion. This “antigenic variation” allows T. brucei to survive in the host blood stream by evading immune response. VSGs arrive at the plasma membrane after entering the secretory pathway at the endoplasmic reticulum (ER) (McConville et al., 2002, Microbiol Mol Biol Rev; 66(1):122-54). The transferrin receptor and nucleobase/nucleoside transporters are other proteins that localize to the plasma membrane after signal peptide dependent import into the ER. Thus, movement of proteins into the ER is crucial for targeting of cell surface receptors and nutrient transporters, as well as for the biogenesis of the Golgi complex, lysosomes, endosomes and the inner nuclear membrane. From the perspective of eukaryotic pathogen control, small molecules that selectively interfere with translocation of proteins into the ER hold promise for treatment of disease because they could block delivery of many proteins to the plasma membrane and as a result compromise viability of parasites. There is a need for efficient, cost-effective assays for identifying drug targets that interfere with protein translocation in trypanosomes.
SUMMARY OF THE INVENTION
The present invention includes a cell free preparation of protozoan microsomes, wherein the cell free preparation of protozoan microsomes translocate a polypeptide into the microsome. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate. In some embodiments, the translocated polypeptide is a secreted polypeptide or a plasma membrane polypeptide. In some embodiments the translocated polypeptide is protected from exogenous detergent.
The present invention includes a method of preparing a cell free preparation of protozoan microsomes wherein the cell free preparation of protozoan microsomes translocate a polypeptide into the microsome. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of endoplasmic reticulum (ER) translocation of a polypeptide using a cell free preparation of protozoan microsomes. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of screening for an agent that modulates the ER translocation of a polypeptide in a protozoan, the method including contacting a cell free preparation of protozoan microsomes with an agent, and monitoring ER translocation of a polypeptide. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of screening for an agent for the treatment and/or prevention of a protozoan infection, the method including contacting a cell free preparation of protozoan microsomes with an agent, and monitoring ER translocation of a polypeptide, wherein a modulation in the ER translocation of the polypeptide indicates the agent is a candidate for the treatment of a protozoan infection. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of screening for an agent that kills, inhibits the growth, and/or inhibits the reproduction of a protozoan, the method including contacting a cell free preparation of protozoan microsomes with an agent, and monitoring ER translocation of a polypeptide, wherein a modulation in the ER translocation of the polypeptide indicates the agent is a candidate agent that kills, inhibits the growth, and/or inhibits the reproduction of a protozoan. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of treating a protozoan infection in a subject, the method including administering to the subject an effective amount of an agent that modulates the ER translocation of a polypeptide in a cell free preparation of protozoan microsomes. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
The present invention includes a method of killing, inhibiting the growth and/or inhibiting the reproduction of a protozoan, the method including contacting the protozoan with an agent that modulates the ER translocation of a polypeptide in a cell free preparation of protozoan microsomes. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
In some embodiments of the methods of the present invention, modulation is a decrease or inhibition in the ER translocation of the polypeptide.
The present invention includes a cell free preparation of protozoan cytosol, wherein the preparation of protozoan cytosol is the supernatant of a high speed spin of greater than 20,000 g of a protozoan lysate, wherein the protozoan cytosol preparation demonstrates the ability to facilitate the translocation of a plasma membrane polypeptide into a cell free preparation of protozoan microsomes. In some embodiments, a high speed spin is greater than 20,000 g. In some embodiments, the cell free preparation of protozoan microsomes is the resuspended pellet of a low sped spin of about 5,000 g to about 20,000 g of a protozoan lysate.
In some embodiments of the methods, cell free preparations, agents, and compositions of the present invention, the protozoan is of the genus Trypanosoma. In some embodiments of the methods, cell free preparations, agents, and compositions of the present invention, the protozoan is T. cruzi, T. brucei, T.b. gambiense, or T.b. rhodesiense. In some embodiments of the methods, cell free preparations, agents, and compositions of the present invention, the protozoan is of the genus Leishmania. 
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B demonstrate cell-free protein import into T. brucei endoplasmic reticulum (ER) membranes. FIG. 1A is a flowchart of protocol for post-translational import of VSG—11786. The various steps and temperatures are depicted. The translation product for VSG—11786 is presented along with [14C] methylated protein markers (Amersham). FIG. 1B shows the import of VSG—11786 into T. brucei microsomes (TbRM). VSG—11786 mRNA was translated in rabbit reticulocyte lysate and then treated with cycloheximide. Reaction mixtures were incubated with TbRM, followed by proteinase K digestion for 60 minutes on ice. Proteins were resolved by SDS-PAGE and detected by phosphorimaging. Lane 1 is untreated VSG—11786; lane 2 is VSG—117500 treated with proteinase K; lane 3 is VSG—11786 translocated in presence of TbRM; lane 4 is VSG—11786 translocated in presence of TbRM but treated with proteinase K; and lane 5 is VSG—11786 translocated in presence of TbRM and then permeabilized with 2% NP40 during proteinase K digestion. Rectangular brackets underneath sets of bars denote those data points that were directly compared for quantitation, and the asterisks denote instances where no proteins were detected.
FIGS. 2A and 2B show the effect of protein translocation inhibitors on TbRM. FIG. 2A presents the protocol used for import of VSG—117500. In FIG. 2B, SG—117500 mRNA was translated in rabbit reticulocyte lysate with 1.5 equivalents T. brucei cytosol (pretreated with MAL3-101 (0.3 μM), MAL3-51 (1 μM), CJ21,058 (20 μM) or equisetin (50 μM)) for 60 minutes. The reaction mixtures were treated with cycloheximide (50 μg/ml, final concentration) and incubated with TbRM (one equivalent). The mixture was incubated for 60 minutes at 37° C. and digested with proteinase K digestion (30 μg/ml, final concentration) for 60 minutes on ice. Proteins were resolved by SDS-PAGE and detected by phosphorimaging. In lanes 1 and 2, TbRM were pretreated with DMSO. Lane 1 is untreated VSG—117500 with TbRM; lane 2 is VSG—117500 incubated with TbRM and treated with proteinase K. In lanes 3 and 4 TbRM were pretreated with MAL3-101. Lane 3 is VSG—117500 incubated with TbRM; lane 4 is VSG—117500 incubated with TbRM and then treated with proteinase K. In lanes 5 and 6 TbRM pretreated with MAL3-51. Lane 5 is VSG—117500 incubated with TbRM; lane 6 is VSG—117500 incubated with TbRM and then treated with proteinase K. In lanes 7 and 8 TbRM were pretreated with CJ-21,058). Lane 7 is VSG—117500 incubated with TbRM; lane 8 is VSG—117500 incubated with ThRM and then treated with proteinase K. In lanes 9 and 10 TbRM were pretreated with equisetin). Lane 9 is VSG—117500 incubated with TbRM; lane 10 is VSG—117500 incubated with TbRM and then treated with proteinase K.
FIGS. 3A to 3D demonstrate the trypanocidal effect of protein translocation blockers (PTBs). Blood stream form T. brucei CA427 were grown in HMI-9 media to the cell density of 106. The cells were then transferred to 96 well plates with the addition of different concentrations of MAL3-101, MAL3-51, CJ-21,058 or equisetin. In controls, DMSO was added. Cell density was calculated at the end of 24 hours, and the graph was plotted. FIG. 3A demonstrates the trypanocidal effect of MAL3-101. FIG. 3B demonstrates the trypanocidal effect of MAL3-51. FIG. 3C demonstrates the trypanocidal effect of CJ-21,058. FIG. 3D demonstrates the trypanocidal effect of equisetin.
FIGS. 4A and 4B demonstrate the effect of anti-trypanosome compounds on a human HeLa Cells. HeLa cells, treated with DMSO, MAL3-101, MAL3-51, CJ-21,058 or equisetin, were subject to a propidium iodide viability assay and analyzed by flow cytometry.
FIGS. 5A to 5D presents the chemical structures of MAL3-101, MAL3-51, CJ-21,058 and Equisetin. FIG. 5A is MAL3-101; FIG. 5B is MAL3-51; FIG. 5C is CJ-21,058; and FIG. 5D is equisetin.
FIGS. 6A to 6D. Structures of MAL3-101, MAL3-51, CAM741, and NFI028. The chemical structures for MAL3-101 (FIG. 6A) and MAL3-51 (FIG. 6B) were reproduced from (Wright et al., 2008, Bioorg Med Chem; 16(6):3291-301), using ChemBioDraw Ultra (version 11.0.1 CambridgeSoft). The pyrimidinone group is highlighted by a box of dotted lines. The peptoid group is highlighted by a box of dashed lines, and substitutions (R1-R4) are marked by brackets. CAM741 (FIG. 6C) and NFI028 (FIG. 6D) chemical structures were reproduced from (Harant et al., 2007, Mol Pharmacol; 71(6):1657-65), using ChemBioDraw Ultra (version 11.0.1 CambridgeSoft). Different spatial orientations between CAM741 and NFI028 are highlighted by light grey boxes, and different chemical substituents are denoted by dark grey boxes.
FIGS. 7A to 7C. MAL3-101 and MAL3-51 affect import of VSG-11786 into T. brucei microsomes. FIG. 7A demonstrates the effects of MAL3-101 and MAL3-51 (10 μM) on VSG-11786 import into T. brucei microsomes in the absence or presence of T. brucei cytosol. VSG-11786 mRNA was translated in rabbit reticulocyte lysate in the absence or presence of T. brucei cytosol for 1 hour at 37° C., and then cycloheximide was added to stop protein synthesis. TbRM (1 equivalent) and MAL3-101 (10 μM), MAL3-51 (10 μM), or an equal volume of DMSO was added to the samples on ice, and the mixture was incubated at 37° C. for 1.5 hours for translocation of VSG-11786 into the microsomes. Reaction mixtures were treated with proteinase K (PK) for one hour on ice. Proteolysis was terminated with PMSF. Proteins were precipitated with ammonium sulfate, pelleted by centrifugation, resuspended in SDS-PAGE sample buffer, and resolved by Tris-Tricine SDS-PAGE. Gel images were obtained with a phosphorimager and analyzed with Quantity-One software. Reaction mixtures for odd numbered lanes contained TbRM, but not proteinase K; even numbered lanes contained TbRM and proteinase K. Brackets denote lanes that were compared to obtain percentage of VSG-11786 import values. FIG. 7B is a graphical representation of data from FIG. 7A. Quantitation of percent VSG import may not correspond to gel image because control lanes (VSG detected in the presence of TbRM without proteinase K) were adjusted to pixel saturation. In FIG. 7C, the effect of MAL3-101 on T. brucei growth.
FIGS. 8A to 8D. Effects of NFI028 and CAM741 on import of VSG-11786 into T. brucei microsomes. FIG. 8A demonstrates the import of VSG-11786 into T. brucei microsomes in the presence of NFI028 or CAM741 (1 μM). VSG-11786 mRNA was translated in rabbit reticulocyte lysate in the presence of T. brucei cytosol for one hour at 37° C., and then protein synthesis was stopped by the addition of cycloheximide. TbRM (1 equivalent) and NFI028 or CAM741 (1 μM) or an equal volume of DMSO was added to the translation mixture on ice, and incubated at 37° C. for 1.5 hours. Reaction mixtures were treated with proteinase K (PK) on ice for one hour. Proteolysis was terminated with PMSF. Proteins were precipitated with ammonium sulfate, pelleted, resuspended in SDS-PAGE sample buffer, and resolved by Tris-Tricine SDS-PAGE. Images of radiolabeled proteins were obtained with a phosphorimager and quantitated with Quantity-One software. Samples depicted in odd numbered lanes contained TbRM, but were not treated proteinase K. Reaction mixtures shown in even numbered lanes contained TbRM and were treated with proteinase K. Brackets denote lanes that were compared to each other in order to obtain percentage of VSG-11786 import values. FIG. 8B is a graphical representation of data from FIG. 8A. FIG. 8C demonstrates the effect of NFI028 on growth of T. brucei. FIG. 8D demonstrates the effect of CAM741 on T. brucei. 
FIGS. 9A to 9D. Effects of azide, AEE788, and valinomycin on VSG-11786 import into T. brucei microsomes. FIG. 9A demonstrates the import of VSG-11786 into TbRM in the presence of sodium azide or AEE788. VSG-11786 mRNA was translated in rabbit reticulocyte lysate in the presence of T. brucei cytosol for one hour at 37° C. Cycloheximide was added to stop proteins synthesis. TbRM (1 equivalent) and sodium azide (8 μM), AEE788 (8 μM), an equal volume of microsome buffer (RMB) or DMSO was added to the samples on ice, and the mixtures were incubated at 37 C for 1.5 hours. Reaction mixtures were treated with proteinase K (PK) on ice for one hour. Proteolysis was terminated with PMSF. Proteins were precipitated with ammonium sulfate, pelleted, resuspended in SDS-PAGE sample buffer, and resolved by Tris-Tricine SDS-PAGE. Images of radiolabeled proteins were obtained with a phosphorimager and quantitated with Quantity-One software. Brackets show lanes that were compared to each other in order to obtain percentage of VSG-11786 imported into TbRM. Lanes 1, 3, 5, & 7 were treated with TbRM, but not proteinase K. Lanes 2, 4, 6, & 8 were treated with TbRM and proteinase K. FIG. 9B is a graphical representation of data from FIG. 9A. FIG. 9C demonstrates VSG-11786 import into TbRM pre-treated with sodium azide, NFI028, or valinomycin was tested using a protocol similar to that described in FIG. 9A, but with the following changes. TbRM that had been pre-incubated for one hour on ice with control buffer (RMB) (lanes 1 and 2), sodium azide (8 μM) (lanes 3 and 4), an equal volume of DMSO (lanes 5 and 6), NFI028 (1 μM) (lanes 7 and 8), valinomycin (0.5 μM) (lanes 9 and 10), or an equal volume of 100% ethanol (lanes 11 and 12) was added to the reaction mixtures and incubated at 37° C. for 1.5 hours. Reaction mixtures depicted in odd lanes contained TbRM, but were not treated with proteinase K; even lanes contained TbRM and were treated with proteinase K. FIG. 9D is a graphical representation of data from panel FIG. 9C.
FIGS. 10A and 10B demonstrate import into T. brucei microsomes and hydrophobicity properties of VSG-11786, (Gly)4-VSG-11786, (Ser)4-VSG-11786, (Ala)7-VSG-11786, (Ala)6-Ile12-VSG-11786, and (Ala)4-VSG-11786 h-Regions. FIG. 6A demonstrates the import of VSG-11786 (“A”), (Gly)4-VSG-11786 (“B”), (Ser)4-VSG-11786 (“C”), (Ala)7-VSG-11786 (“D”), (Ala)6-Ile12-VSG-11786 (“E”), and (Ala)4-VSG-11786 (“F”) into T. brucei microsomes. mRNAs of VSG-117 variants truncated to 86 amino acids were translated in rabbit reticulocyte lysate. Translation was stopped with cycloheximide, and the translation mixture was supplemented with T. brucei microsomes (TbRM), allowing translocation to occur for 1.5 hours at 37° C. Translocation was stopped by incubation of the sample on ice, and import of the protein was tested with proteinase K (PK) digestion on ice for one hour. The samples were precipitated with ammonium sulfate, resolved on Tris-Tricine gels, and analyzed with a phosphorimager and Quantity One software. Gels pictured are representative results. In FIG. 10A (panels “A”-“F”), Lane 1 is no TbRM, no PK; lane 2 is no TbRM, with PK; lane 3 is with TbRM, no PK; and lane 4 is with TbRM, with PK. Brackets denote lanes that were compared to obtain percentage of VSG imported into TbRM. In FIG. 10B peak hydrophobicity values (black bars) were obtained by Kyte-Doolittle hydrophobicity analysis (window=7 amino acids), using LASERGENE (DNASTAR) (version 4.0.3). Percentage of VSG imported (gray bars) from data in FIG. 10A, panels “A”-“F,” was calculated by dividing the amount of the residual VSG detected after protease treatment (lane 4) by the amount of the VSG detected after the addition of TbRM with no protease treatment (lane 3) and multiplying the quotient by 100. Quantitation of percent VSG import may not correspond to gel image because control lanes (VSG detected in the presence of TbRM without proteinase K) were adjusted to pixel saturation.
FIGS. 11A and 11B present translocation time courses of VSG-11786 and (Ala)4-VSG-11786 into T. brucei microsomes. FIG. 11A panel “A” is a time course of importing VSG-11786 into TbRM and FIG. 11A panel “B” is a time course of importing (Ala)4-VSG-11786 into TbRM. mRNAs of VSG-117's truncated to 86 amino acids were translated in rabbit reticulocyte lysate. Translation was stopped with cycloheximide, and the translation mixture was supplemented with TbRM and incubated at 37° C. In FIG. 11A, translocation was allowed to occur for 0.25 hours (lanes 1 and 2), 0.5 hours (lanes 3 and 4), 1.0 hour (lanes 5 and 6), 1.5 hours (lanes 7 and 8), or 2.0 hours (lanes 9 and 10). Translocation was stopped by incubating the sample on ice. VSG import into TbRM was tested with proteinase K (PK) digestion on ice for one hour. Reaction mixtures were precipitated with ammonium sulfate, resolved on Tris-Tricine gels, and analyzed with a phosphorimager and Quantity One software. Gels pictured are representative results. Lanes 1, 3, 5, 7, and 9 are TbRM present, no PK treatment; lanes 2, 4, 6, 8, and 10 are TbRM present, PK present. Brackets denote lanes that were compared to obtain percentage of VSG imported into TbRM. FIG. 11B is a graphical representation of data in panels A and B of FIG. 11A. Percentage of VSG imported was calculated by dividing the amount of the residual VSG detected after protease treatment by the amount of the VSG detected after the addition of ThRM without protease treatment, and multiplying the quotient by 100.
FIGS. 12A and 12B demonstrate that the rearrangement of h-region sequences affects VSG translocation into TbRM mRNAs of VSGs translated in rabbit reticulocyte lysate. Translation was stopped with cycloheximide. TbRM was added. The mixtures were incubated at 37° C. for 1.5 hours. Import of VSG-11786 into TbRM was tested by proteinase K (PK) digestion on ice for one hour. Proteins in the reaction mixture were precipitated with ammonium sulfate, resolved on Tris-Tricine gels, and analyzed with a phosphorimager and Quantity One software. Gels pictured are representative results. In FIG. 12A panels “A”-“F,” Lane 1 is no TbRM, no PK; lane 2 is no TbRM, with PK; lane 3 is with TbRM, no PK; lane 4 is with TbRM, with PK. Brackets denote lanes that were compared to obtain percentage of VSG imported into TbRM for each study. h-Regions studied in FIG. 12A were VSG-11786 (“A”), (Ala)4-VSG-11786 (“B”), scrambled (Ala)4-VSG-11786 (“C”), (Ser)4-VSG-11786 (“D”), scrambled (Ser)4-VSG-11786 (“E”), and (Ala)4-Tyr9-VSG-11786 (“F”). FIG. 12B presents peak hydrophobicity values (black bars) obtained by Kyte-Doolittle hydrophobicity analysis (window=7 amino acids), using LASERGENE (DNASTAR) (version 4.0.3). Percentage of VSG imported into TbRM (gray bars) are from data in panels A-F; it was calculated by dividing the amount of the residual VSG detected after protease treatment by the amount of the VSG detected after addition of ThRM without protease treatment, and multiplying the quotient by 100.
FIGS. 13A and 13B demonstrate microsomal import and hydrophobicity properties of VSG-11786, (Phe)4-VSG-11786, and (Val)4-VSG-11786. FIG. 13A presents import of VSG-11786 (“A”), (Phe)4-VSG-11786 (“B”), and (Val)4-VSG-11786 (“C”) into T. brucei microsomes. mRNAs of VSG-117 variants were translated in rabbit reticulocyte lysate. Translation was stopped with cycloheximide. The reaction mixtures were supplemented with TbRM, and incubated at 37° C. for 1.5 hours. Translocation into TbRM was stopped by incubation of the samples on ice, and import of VSG was tested by proteinase K (PK) digestion on ice for one hour. The samples were precipitated with ammonium sulfate, proteins were resolved on Tris-Tricine gels, and analyzed with a phosphorimager and Quantity One software. Gels pictured are representative results. In FIG. 13A panels “A”-“C,” Lane 1 is no TbRM, no PK; lane 2 is no TbRM, with PK; lane 3 is with TbRM, no PK; lane 4 is with TbRM, with PK. Brackets denote pairs of lanes that were compared to obtain percentage of VSG imported into TbRM in each set. In FIG. 13B peak hydrophobicity values (black bars) were obtained by Kyte-Doolittle hydrophobicity analysis (window=7 amino acids), using LASERGENE (DNASTAR) (version 4.0.3). Percentage of VSG imported (gray bars) was calculated from data in FIG. 13A panels A-C by dividing the amount of the residual VSG detected after protease treatment by the amount of the VSG detected after addition of TbRM without protease treatment, and multiplying the quotient by 100.
FIGS. 14A and 14B demonstrate the effect of replacing leucine at h-region position six with phenylalanine, valine, or isoleucine on the translocation of VSG into TbRM. FIG. 14A demonstrates the import of VSG-11786 (“A”), (Ala)4-VSG-11786 (“B”), (Ala)4-Phe6-VSG-11786 (“C”), (Ala)4-Val6-VSG-11786 (“D”), and (Ala)4-Ile6-VSG-11786 (“E”) into T. brucei microsomes. mRNAs of VSG-117 were translated in rabbit reticulocyte lysate. Translation was stopped with cycloheximide, and the translation mixture was supplemented with TbRM, followed by incubation at 37° C. for 1.5 hours. Translocation was stopped by incubation of the reaction mixture on ice, and import of proteins was tested by proteinase K (PK) digestion on ice for one hour. Proteins were precipitated with ammonium sulfate, resolved on Tris-Tricine gels, and analyzed with a phosphorimager and Quantity One software. Gels pictured are representative results. In FIG. 14A panels “A”-“E,” Lane 1 is with TbRM, no PK; and lane 2 is with TbRM, with PK. Brackets denote pairs of lanes that were compared in order to obtain percentage of VSG imported into TbRM. In FIG. 14B peak hydrophobicity values (black bars) were obtained by Kyte-Doolittle hydrophobicity analysis (window=7 amino acids), using LASERGENE (DNASTAR) (version 4.0.3). Percentage of VSG imported (gray bars) was calculated from data in FIG. 14A panels A-E by dividing the amount of the residual VSG detected after protease treatment by the amount of the VSG detected after addition of TbRM without protease treatment, and multiplying the quotient by 100.
FIG. 15 presents a helical wheel analysis of VSG-117 h-region. The helical wheel model of VSG-117 h-region was generated with LASERGENE (DNASTAR) (Protean Module, version 4.0.3). Arrows indicate h-region “identity components” (IC1, IC2, IC3, IC4, and IC5) in the VSG-117 h-region (see Table 3).
FIGS. 16A to 16D present models for Sec61 and signal peptide h-region interactions. FIG. 16A is a top view of a VSG-117 signal peptide embedded within a T brucei Sec61 translocon model. FIG. 16B is a top view. FIG. 16C is a side view of a VSG-117 signal peptide placed between TM2b and TM7 of T. brucei Sec61α (TbSec61α). FIG. 16D shows T. brucei VSG-117 h-region α-helix interacting with TM2b and TM7 of T. brucei Sec61α(not drawn to scale). It is predicted that the h-region of a VSG signal peptide forms an α-helix, and uses h-motif identity components to interact with TM2b and TM7 of TbSec61α. Side-chains of “identity components” of the h-motif are positioned to interact with TM2b and/or TM7 of TbSec61α. The h-motif “identity components” displayed here are from VSG-117 (L4, L6, L8, L9, and A11), and the TbSec61 translocon is a “homology model” of the T. brucei Sec61 translocon based on the X-Ray crystal structure from Methanococcus jannaschii (Van den Berg et al., 2004, Nature; 427:36-44).
FIGS. 17A to 17D demonstrates cell-free protein import into T. brucei ER membranes. FIG. 17A is a flow chart of the protocol for post-translational Import of full length VSG. Depicted are the various steps, duration and temperatures at which reactions took place. FIG. 17B demonstrates the import of full length VSG—117 into TbRM. VSG—117 mRNA was translated in rabbit reticulocyte lysate for 15 minutes and then cycloheximide (50 μg/ml, final concentration) was added. Cytosol from T. brucei was also added to all reactions. Reaction mixtures were incubated with TbRM (one equivalent) for 45 minutes (37° C.) followed by proteinase K digestion (300 μg/ml, final concentration) on ice for 60 minutes. Proteins were resolved by SDS-PAGE and detected by phosphorimaging. Lane 1 is VSG—117 translated in reticulocyte lysate; lane 2 is VSG—117 digested with proteinase K; lane 3 is VSG—117 digested with proteinase K in the presence of NP-40 (2%); lane 4 is VSG—117 incubated with TbRM; lane 5 is VSG—117 incubated with TbRM and digested with proteinase K; lane 6 is VSG—117 translocated into TbRM and then permeabilized with 2% NP-40 during proteinase K digestion. FIG. 17C demonstrates translocation of VSG_MVAT7 into TbRM. Lane 1 is VSG_MVAT7 translated in reticulocyte lysate; lane 2 is VSG_MVAT7 produced in reticulocyte lysate and digested with proteinase K. Lane 3 is VSG_MVAT7 incubated with TbRM; lane 4 is VSG_MVAT7 incubated with TbRM and then challenged with proteinase K; lane 5 is VSG_MVAT7 incubated with TbRM, permeabilized with NP-40 (2%), and digested with proteinase K. FIG. 17D demonstrates that a signal peptide is required for VSG—117500 ΔSP translocation. VSG—117500ΔSP mRNA was translated in a rabbit reticulocyte lysate with T brucei cytosol (1.5 equivalents) for 60 minutes, and treated with cycloheximide (50 μg/ml, final concentration). VSG—117500ΔSP was incubated with TbRM (one equivalent) for 60 minutes at 37° C. and digested with proteinase K (120 μg/ml, final concentration) for 60 minutes on ice. Proteins were separated by SDS-PAGE and radiolabeled polypeptides were detected by phosphorimaging. Lane 1 is untreated VSG—117500ΔSP; lane 2 is VSG—117500ΔSP treated with proteinase K; lane 3 is VSG—117500ΔSP incubated with TbRM; lane 4 is VSG—117500ΔSP incubated with TbRM and treated with proteinase K. Rectangular brackets underneath sets of bars denote those data points that were directly compared for quantification, and the asterisks denote instances where no signal was detected.
FIGS. 18A and 18B. MAL3-101, equisetin, and CJ-21,058 inhibit VSG translocation into TbRM. FIG. 18A presents the protocol used for import of VSG—117. In FIG. 18B, VSG—117 mRNA was translated in rabbit reticulocyte lysate with 1.5 equivalents T. brucei cytosol pretreated with MAL3-101 (3 μM), MAL3-51 (10 μM), CJ-21,058 (20 μM) or equisetin (50 μM)) for 60 minutes. The reaction mixtures received cycloheximide (50 μg/ml, final concentration) and were incubated with TbRM (one equivalent). After incubation at 37° C. for 60 minutes, reaction mixtures were transferred to an ice-water bath and treated with proteinase K (30 μg/ml, final concentration) for 60 minutes. Proteins were resolved by SDS-PAGE and detected by phosphorimaging. Lane 1 is VSG—117 incubated with TbRM; lane 2 is VSG—117 digested with proteinase K after incubation with TbRM; lane 3 is VSG—117 incubated with MAL3-101 in the presence of TbRM; lane 4 is VSG—117 and TbRM incubated with MAL3-101 and then digested with proteinase K; lane 5 is VSG—117 incubated with MAL3-51 and TbRM; lane 6 is VSG—117 incubated with TbRM in the presence of equisetin; lane 7 is VSG—117 incubated with ThRM; lane 8 is VSG—117 incubated with equisetin and digested with proteinase K; lane 9 is VSG—117 incubated with CJ-21,058 and TbRM; lane 10 is VSG—117 incubated with TbRM and CJ-21,058 and digested with proteinase K. For quantitation, each proteasedigested sample was compared to a control experiment containing the drug but without protease addition. Thus, lane 2 is compared with lane 1 while lane 4 is compared to lane 3, and so on.
FIGS. 19A to 19D. VSG—117 bound to ThRM is Resistant to Carbonate Extraction and Floats on a Sucrose Cushion. FIG. 19A presents SDS-PAGE of carbonate extraction of VSG-117 in the absence or presence of TbRM. Lanes 1 and 2 represent VSG-117 carbonate extraction without TbRM. Lanes 3 and 4 represent VSG-117 carbonate extraction with TbRM present. “S” denotes proteins precipitated from the supernatant of the 230,000×g step while “P” represents proteins recovered from the 230,000×g centrifugation step. Brackets denote lanes that were compared to obtain a percentage to total VSG-117 present. FIG. 19B is a quantitation of data in FIG. 19A; the percentage of total VSG-117 present is expressed as the amount of VSG detected in a single lane divided by the sum of the amount of VSG detected in the supernatant and pellet lanes. FIG. 19C presents Tris-Tricine SDS-PAGE after he floatation of VSG-117 in the absence (top panel) or presence (bottom panel) of TbRM. Lane 1 represents proteins obtained from the 0.25 M sucrose layer; lane 2 represents proteins obtained from the 1.5 M sucrose layer; lane 3 represents proteins obtained from the top 2.3 M sucrose layer; and lane 4 represents proteins recovered from the bottom 2.3 M sucrose layer and those protein pelleted from the 100,000×g centrifugation step. FIG. 19D is a quantitation of the data in FIG. 19C. Percentage of total VSG-117 present is expressed as the amount of VSG detected in a single lane divided by the sum of VSG detected in lanes 1-4. “*” Represents 0% VSG-117 detected.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The present invention provides cell free preparations of protozoan microsomes, wherein the cell free preparation of protozoan microsomes demonstrate the ability to translocate a protozoan polypeptide into the microsome, methods of preparing cell free preparations of protozoan microsomes, and methods of using cell free preparations of protozoan microsomes. As used herein, a microsome is a small vesicle that is derived from fragmented endoplasmic reticulum (ER). Microsomes may be prepared from a lysate of protozoan cells. Microsomes may be purified and isolated from other cellular components, such as, for example, unbroken cells, other cellular organelles, nuclei, mitochondria, soluble enzymes, and/or other soluble proteins of the cellular cytosol.
The protozoan microsomes of the present invention may be prepared from any of a variety of protozoa, including, for example, kinetoplastid protozoa, such as for example, protozoa of the Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leishmania, Leptomonas, Phytomonas, Trypanosoma, and Wallaceina genera. In preferred embodiments, the protozoan is of the genus Trypanosoma, including, but not limited to, T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense. In some embodiments, the protozoan is of the genus Leishmania, including, for example, Leishmania major. Notable trypanosomal diseases include trypanosomiasis (African Sleeping Sickness and South American Chagas Disease, caused by species of Trypanosoma) and leishmaniasis (caused by species of Leishmania).
The purified and isolated microsomes of the present invention demonstrate the capability of translocating polypeptides across the endoplasmic reticulum (ER). In some embodiments, the polypeptide is a polypeptide of protozoan origin or is a polypeptide of non-protozoan origin, with a translocation signal sequence of protozoan origin. Translocated polypeptides may be, for example, plasma membrane polypeptides or soluble polypeptides. Translocation of a polypeptide into the microsome may be assayed by any of a variety of methods, including, but not limited to, protection of the translocated polypeptide from proteinase, protection of the polypeptide from endogenous detergent, and/or loss of protease protection after detergent permeabilization of the microsome.
To prepare cell free microsomes, protozoan cells may be obtained by any of the many methods known to the skilled artisan. Protozoan cells may be treated in a manner to disrupt the protozoan cells, so that the protozoan cell membrane is broken open. This process is known as homogenization of the protozoan cell or preparation of a protozoan cell lysate. Protozoan cells may be homogenized by any of a variety of methods, including, but not limited to, osmotic alteration, mechanical disruption, sonification, enzymatic digestion, detergent-based cell lysis, or a combination of two or more of these methods. Methods of mechanical disruption include, but are not limited to, dounce homogenization, mortars, pestles, nitrogen burst methods, french pressure cell press and pumped-fluid processors.
Microsomes may be concentrated and separated from other cellular organelles by differential centrifugation of a protozoan cell lysate to sediment out unbroken cells, nuclei, mitochondria, and other cellular organelles, whereas soluble cytosolic proteins and microsomes remain in the supernatant. Such centrifugation may be a low speed centrifugation at a speed of less than about 5,000 g, including, but not limited to, for example, about 1,000 g, about 2,000 g, about 3,000 g, about 4,000 g, about 5,000 g, or less, or about 2,000 to about 4,000 g.
Microsomes may be separated from soluble cytosolic proteins by differential centrifugation to sediment out the microsomes formed by fragments of ER membrane, whereas the soluble cytosolic proteins remain in the supernatant and the microsomes are found in the pellet. Such microsomes may be pelleted by a speed of about 5,000 g to about 20,000 g. Such microsomes may be pelleted at a speed of about 5,000 g, about 10,000 g, or about 12,000 g to about 14,000 g, about 16,000 g, about 18,000 g, or about 20,000 g. The pelleted microsomes may be resuspended, for example, in a resuspension solution. Such a buffer may be isotonic and/or pH buffered.
Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures and is widely used in industry and laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid.” Protocols for centrifugation typically specify the amount of acceleration to be applied to the sample or a rotational speed such as revolutions per minute (rpm). The acceleration is often quoted in multiples of “g,” the acceleration due to gravity at the Earth's surface. This distinction is important because two rotors with different diameters running at the same rotational speed will subject samples to different accelerations. The acceleration can be calculated as the product of the radius and the square of the angular velocity using the formula:

g=RCF=0.00001118×r×N2,

where r=rotational radius (centimeter, cm)

and N=rotating speed (revolutions per minute, r/min). To avoid having to perform a mathematical calculation every time, nomograms for converting RCF to rpm for a given rotor of a given radius are widely available.

The present invention also includes a purified cytosol preparation of the supernatant from which microsomes have been removed. This cytosol preparation may be subject to additional centrifugation and/or purification procedures. For example, such a cytosol preparation may the supernatant of a high speed of greater than 20,000 g, including, but not limited to, speeds of about 65,000×g, about 75,000 g, about 100,000 g, about 120,000 g, about 180,000 g, and about 200,000 g. This cytosol preparation may include purified soluble cytosolic proteins that facilitate the translocation of a polypeptide across a microsome.
The present invention includes any of the microsomes, methods of making microsomes, and methods of making cytosol preparations described herein, including, but not limited to, any of those described in the Examples included herewith.
For example, T. brucei microsomes, also referred to herein as “TbRM,” may be prepared by obtaining blood stream trypanosomes from the blood of infected rats. Trypanosomes may be mechanically broken by homogenization, for example, with a dounce homogenizer. A homogenization buffer of about 250 mM Sucrose, about 50 mM HEPES-KOH, about 50 mM KOAc, about 6 mM Mg(OAc)2, about 1 mM EDTA, about 1 mM DTT, about 1 μg/ml TLCK, about 5 μg/ml leupeptin, and about 0.5 mM PMSF (final concentrations) may be used. The homogenized trypanosome lysate may be centrifuged in a low speed spin of less than about 5,000 g to remove unbroken cells, nuclei, mitochondria, and other cellular organelles. The supernatant of the low speed spin, containing the protozoan microsomes may then be centrifuged at a higher speed, for example, about 5,000 to about 20,000 g, to pellet the microsomes. Microsomes may be resuspended in, for example, rough microsome buffer (RMB) of about 250 mM Sucrose, about 50 mM HEPES-KOH, about 50 mM KOAc, about 6 mM Mg(OAc)2, about 1 mM DTT, about 0.5 μg/ml TLCK, and about 2.5 μg/ml leupeptin. Microsome concentration may be determined, for example, by measuring absorbance at 260 nm. Concentration of microsomes may be adjusted (with, for example, RMB) to OD260 nanometers (nm) of 50. In some aspects, one equivalent of TbRM has an OD260 of 50 nm.
The present invention also includes a purified cytosol preparation of the supernatant from which microsomes have been removed. This cytosol preparation may be subject to additional centrifugation and/or purification procedures. For example, the supernatant obtained during the preparation of TbRM described above may be centrifuged at a speed greater than 20,000 g. The resulting supernatant may be further concentrated by ultrafiltration. Concentration of the cytosol preparation may be adjusted (with 0.1% SDS) to OD280 nanometers (nm) of 50. In some aspects, one equivalent of cytosol has an OD280 of 50 nm. The cytosol preparation may be quick-frozen in liquid nitrogen, and stored at −80° C. Such purified cytosol preparations may be added to a cell free microsome preparation to increase, improve or facilitate the translocation of polypeptides. The translocation of larger polypeptides may, in particular, be enhanced by the addition of such a cytosol preparation.
Signal peptides for translocation across the endoplasmic reticulum are essential for protein traffic through the secretory system in eukaryotes, including protozoans. In addition, lumenal proteins of the ER, Golgi complex, lysosomes and plasma membrane proteins with exoplasmic domains depend on signal peptides for correct intracellular targeting. Parasite surface proteins are critical for the uptake of nutrients, such as, for example, iron, signal transduction, and cell viability. Therefore, an understanding of how proteins arrive at the plasma membrane of a protozoan is important for understanding viability and virulence in these organisms.
In T. brucei, an eukaryote that causes human African trypanosomiasis, the defining features of signal peptides are not known and the ER protein translocation machinery has not been fully characterized. Signal peptides of T brucei are not compatible with the canine microsomal protein import system (Al-Qahtani et al., 1998, Biochem J; 331:521-529 and Ramirez et al., 1999, J Eukaryot Microbiol; 46(6):557-65) that is widely used to study translocation of proteins into eukaryote ER (Connolly et al., 1989, J Cell Biol; 108(2):299-307). For example, in a canine microsomal system, signal peptides from T. brucei, namely VSG—117, VSG—221, VSG_MVAT7 and BiP, fail to direct ER import of proteins. In control experiments, pp MF and E. coli β-lactamase were imported into the canine microsomes. Replacement of the signal sequence of VSG—117 with a signal sequence from pp MF led to import of the T. brucei protein by canine microsomes. Thus, the trypanosome signal peptide is incompatible with the canine ER protein import machinery. Further, trypanosomatid proteins expressed in human and insect cells are mistargeted. Attempts to secrete recombinant L. major gp63 from insect Sf9 cells failed. When the trypanosomatid signal sequence was replaced with a baculovirus signal peptide, gp63 was secreted. When gp82 from T. cruzi was expressed in murine Vero cells the protein was not targeted to the plasma membrane where it is normally found in the parasite. Replacement of the T. cruzi signal peptide with a signal sequence from influenza virus hemagglutinin targeted gp82 to the plasma membrane of Vero cells. Thus, both in vitro and in vivo, trypanosomatid signal peptides seem to be incompatible with ER protein translocation systems in other eukaryotes.
The cell free microsome preparation of the present invention may be used in methods translocating protozoan polypeptides across the microsomal ER membrane. The diversity and nonequivalence of signal sequences in vertebrate is receiving attention (Martoglio and Dobberstein, 1998, Trends Cell Biol; 8(10):410-5; Rutkowski et al., 2003, J Biol Chem; 278(32):30365-72; Hegde and Bernstein, 2006, Trends Biochem Sci; 31(10):563-712006; Harant et al., 2006, J Biol Chem; 281(41):30492-502; and Besemer et al., 2005, Nature; 436(7048):290-3). Since trypanosome and vertebrate signal sequences and ER translocation mechanisms have intrinsic differences (Al-Qahtani et al., 1998, Biochem J; 331:521-529), the T. brucei ER protein import pathway provides a target for drug discovery.
The novel cell free microsome system for trypanosome ER protein import of the present invention is prepared from membranes from T. brucei (TbRM) and is able, unlike canine microsomes, to import proteins with trypanosome signal peptides. Thus, the cell free microsystem of the present invention provides for the identification and characterization of T. brucei signal sequences and other protozoan species. The present invention includes polypeptides including T brucei signal sequences, including, but not limited to, any of the peptide sequences set forth in Example 7. Protein translocation of secretory proteins into the endoplasmic reticulum (ER) in eukaryotes enables polypeptide entry into the secretory pathway, which is important for the viability of the human protozoan parasite Trypanosoma brucei. An N-terminal signal peptide of precursor proteins is essential for entry into the ER. A hydrophobic core, or h-region, of the signal peptide is required for signal peptide activity. h-Regions are thought to be composed of random hydrophobic amino acids, whose peak hydrophobicity governs activity of signal peptides. Example 7 re-evaluated these concepts using a trypanosome microsomal system for import of a variant surface glycoprotein, VSG-117 and found that h-region peak hydrophobicity alone does not dictate signal peptide activity. Moreover, this example identified conserved tri-component peptide motifs (including, L-L-x-[AILV], L-x(1,2)-L-[AILV], and L-x(2,3)-L-[AILPV]) in h-regions of T. brucei signal peptides.
The cell-free microsome translocation system of the present invention can be used to identify candidate drugs for the treatment and prevention of diseases caused by protozoa. As described in the examples included herewith, several small molecules, including two natural compounds of fungal origin, CJ-21,058 (Sugie et al., 2002, J Antibiot (Tokyo); 55(1):25-9) and equisetin, a SecA inhibitor (Vesonder et al., 1979, J Antibiot (Tokyo); 32(7):759-61), block import of a VSG polypeptide into TbRM, and killed T. brucei. Since trypanosome post-translational ER protein import may require molecular chaperones, inhibitors of Hsp70 were also tested. MAL3-101 (Fewell et al., 2004, J Biol Chem; 279(49):51131-40) blocked protein translocation into TbRM and was trypanocidal at sub-micromolar concentrations. These studies establish the ER protein import pathway as a valid target for discovery of anti-trypanosome drugs, and presents the cell free microsome system of the present invention as system for performing focused screens of trypanocidal compounds that inhibit protein import into the ER.
The cell free microsome system of the present invention may be used for the screening and identification of agents for the treatment and/or prevention of a protozoan infection. Such cell free microsome systems may be used for the screening and identification of agents that kill, inhibit the growth, and/or inhibit the reproduction of a protozoan. For example, one may find new anti-trypanosome compounds by either screening chemical libraries or by rational drug design. In rational drug design, one aims to find small molecules that inhibit the function of proteins that are essential for parasite viability (McKerrow, 1996, Biologicals; 24(3):207-8; Barrett et al., 1999, Trends Microbiol; 7(2):82-8; Hammarton et al., 2003, Prog Cell Cycle Res; 5:91-101; Seizer et al., 1999, Proc Natl Acad Sci USA; 96(20):11015-22; Naula et al., 2005, Biochim Biophys Acta; 1754(1-2):151-9; and Gelb et al., Mol Biochem Parasitol; 126(2):155-63).
Assaying for such agents can include one or more of the following: preparing a microsome preparation, preparing a cytosol preparation, contacting a microsome preparation and/or a cytosol preparation with an agent, determining translocation into a microsome in the presence and absence of an agent, and/or evaluating the functional effects of the agent on activity. A wide variety of methods may be used to evaluate translocation, including, but not limited to, any of those described herein.
The cell free protozoan microsomes of the present invention demonstrate the capability of translocating polypeptides across the ER. As used herein, the term “polypeptide” refers to a polymer of amino acids linked by peptide bonds and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. A polypeptide may be, for example, an intact protozoan polypeptide, such as, for example, an intact VSG polypeptide, a truncated protozoan polypeptide, such as, for example, a truncated VSG polypeptide (including, for example, any of those described in the examples included herewith), a chimeric polypeptide, containing, for example, amino acid residues from two different species, such as, for example, trypanosome and human amino acid sequences, and altered polypeptides, in which selected amino acid residues have been changed, for example, by site directed mutagenesis.
As used herein, the term isolated means that a preparation that is either removed from its natural environment or synthetically derived, for instance by recombinant techniques, or chemically or enzymatically synthesized. An isolated polynucleotide denotes a polynucleotide that has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. When applied to a protein/polypeptide, the term isolated indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins.
The present invention includes agents that modulate ER translocation in a cell free protozoan microsome preparation. The present invention includes derivatives and analogs of such agents. As used herein the term “agent” includes both protein and non-protein moieties. In one embodiment, the agent is a small molecule. The agent may be derived from a library of low molecular weight compounds or a library of extracts from plants or other organisms. Such agents and derivatives and analogs thereof may be identified by any of the methods discussed herein. The present invention includes compositions of one or more such agents and/or derivatives and analogs thereof. A composition may be a pharmaceutical composition. A composition may include a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable,” as used herein, means that the compositions or components thereof so described are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, and the like. As used herein in vitro is in cell culture, ex vivo is a cell that has been removed from the body of a subject, and in vivo is within the body of a subject.
The present invention includes methods of treating or preventing a protozoan infection in a subject by administering to the subject an effective amount of an agent that modulates the ER translocation of a polypeptide in a cell free preparation of protozoan microsomes. Such an agent may be identified by the methods described herein. As used herein “treating” or “treatment” includes both therapeutic and prophylactic treatments. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The present invention includes methods of treating or preventing a protozoan infection in a subject by administering to the subject an effective amount of MAL3-101, CJ-21,058, AEE788, equisetin, CAM741, NF 1028, and/or derivatives thereof.
The present invention includes methods of killing, inhibiting the growth of, and/or inhibiting the reproduction of a protozoan by contacting the protozoan with an agent modulates the ER translocation of a polypeptide in a cell free preparation of protozoan microsomes. Such an agent may be identified by any of the methods described herein. A modulation of ER translocation includes a reduction in the rate of ER translocation, an inhibition of ER translocation. In some aspect, a modulation of ER translocation is the halting or prevention of ER translocation. In some aspect, a modulation of ER translocation is an increase in the rate of ER translocation, an enhancement in ER translocation. As used herein, the term “inhibit” means prevent, decrease, or reverse. In some embodiments, modulation is an increases, activation, or stimulation of ER translocation. The present invention includes methods of killing, inhibiting the growth of, and/or inhibiting the reproduction of a protozoan by contacting the protozoan with MAL3-101, CJ-21,058, AEE788, equisetin, CAM741, NF 1028, and/or derivatives thereof.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
EXAMPLES
Example 1
Preparation of T. brucei Microsomes
To prepare T. brucei microsomes (TbRM), blood stream trypanosomes were obtained from the blood of infected rats, and purified by DE-52 chromatography. Cells (1×1010) were resuspended in 5 milliliter (ml) of homogenization buffer (made up of 250 mM Sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1 μg/ml TLCK, 5 μg/ml leupeptin, 0.5 mM PMSF (final concentrations). Two and half ml of resuspended cells were added to pre-chilled dounce homogenizer where they were lysed with 80 strokes of a pestle (specially selected for its tight-fitting to the homogenizer) on ice. After a one minute rest, another after 80 strokes was administered with the pestle. Homogenized cells were recovered and kept on ice while the remaining 2.5 ml of resuspended cells were being broken. Both sets of homogenized lysates were pooled and centrifuged (2,000 rpm, 10 min, 4° C.) in microfuge tubes. The supernatants were pooled, aliquoted into new microfuge tubes, and centrifuged at 12,000 rpm for 20 min at 4° C., and each pellet was resuspended in 50 μl (total volume) of fresh rough microsome buffer (RMB) (250 mM Sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM DTT, 0.5 μg/ml TLCK, 2.5 μg/ml leupeptin). The T. brucei microsome (TbRM) concentration was determined by measuring absorbance at 260 nm. Concentration of TbRM was adjusted (with RMB) to OD260 nanometers (nm) of 50, aliquoted, quick frozen in liquid nitrogen, and stored at −80° C. One equivalent of TbRM has an OD260 of 50 μm. Supernatant from the 12,000 rpm centrifugation was also saved.
Various unique properties distinguish T. brucei from canine pancreas microsomal system. Protein import into the endoplasmic reticulum has been studied extensively in vitro using a canine microsomal system (reviewed in Martoglio and Dobberstein, 1996, Trends Cell Biol; 6:142-147 and Walter and Blobel, 1983, Methods Enzymol; 96:84-93). Canine microsomes import protein co-translationally. They cannot import pre-synthesized protein. Consequently, there is no hard evidence that canine ER protein import requires cytoplasmic chaperones (such as, for example, Hsp70 and Hsp40). Finally, canine microsomal import system cannot recognize trypanosome signal peptides (Al-Qahtani et al., 1998, Biochem J; 331:521-529).
In contrast to the canine microsomal system, T. brucei microsomes (ThRM) of the present invention depend on trypanosome signal peptides to translocate protein into the endoplasmic reticulum (ER). Further, import of protein into TbRM can occur post-translationally, unlike the canine pancreatic system. Finally, translocation of full-length protein into TbRM requires cytosol from the parasite, and is inhibited by a small molecule that interferes with Hsp70 function, suggesting that Hsp40 is crucial for import of protein into TbRM.
Example 2
Endoplasmic Reticulum Protein Import as a Target for Anti-Trypanosome Drug Discovery
Caused by Trypanosoma brucei, human African trypanosomiasis is an emerging disease for which new drugs are needed. As an extracellular parasite in its vertebrate host, expression of proteins, including variant surface glycoprotein (VSG) on the plasma membrane is crucial for establishment and maintenance of an infection. Transport of the majority of proteins to the plasma membrane involves translocation into the endoplasmic reticulum (ER). Prevention of protein import into the parasite ER would be an effective target for the discovery of new anti-trypanosome compounds. In this example, a microsomal system that imports a model VSG—117 (VSG—11786) substrate was developed. This in vitro system was used in a focused screen to identify small molecules that block import of proteins into the microsomes from T. brucei (TbRM). Protein translocation blockers were then tested for anti-trypanosome effects. MAL3-101, an inhibitor of Hsp40-regulated Hsp70 ATPase, blocked import of VSG—11786 into TbRM. Similarly, equisetin and CJ-21,058 also inhibited import of VSG—11786 into TbRM. In tests of trypanocidal activity, all compounds that blocked protein import into TbRM were toxic to trypanosomes. The concentrations at which fifty percent of parasites were killed (IC50) were 125 nM for MAL3-101, 3.3 μM for equisetin, and 7 μM for CJ-21,058. MAL3-101 and equisetin did not have discernible effects on human HeLa cells at concentrations that killed T. brucei. These observations establish TbRM protein import as a rapid assay for anti-trypanosome drug discovery. Drugs, including small molecule drugs, may be screened in the TbRM protein import system of the present invention to identify those that inhibit protein import into the microsomes and potentially kill the parasite. The TbRM protein import system of the present invention will identify lead compounds for anti-trypanosome drug discovery. TbRM protein import system of the present invention has identified MAL3-101, equisetin, and CJ-21,058 as lead compounds for anti-trypanosome drug discovery.
Materials and Methods
Reagents and chemicals. Plasmid pVSG—117 (Bangs, et al., 1986, J Cell Biol; 103:255-263) was provided by Dr. J. Bangs (University of Wisconsin, Madison). CJ-21,058 and equisetin were gifts from Pfizer Inc., (New York, N.Y.). Rabbit reticulocyte lysate (RRL) and methionine/cysteine-free amino acid mixture was purchased from Promega (Madison, Wis.). [35S]Redivue PromixTM was purchased from Amersham Biosciences (Piscataway, N.J.), etoposide was purchased from Sigma (St. Louis, Mo.), and propidium iodide was purchased from Invitrogen (Carlsbad, Calif.). AmpliscribeTM T7 in vitro transcription kit was purchased from Epicentre Technologies (Madison, Wis.).
DNA Templates and RNA Synthesis in vitro. DNA template (one μg) was transcribed using the AmpliscribeTM T7 kit (Epicentre Technologies) following the manufacturer's protocol. A template for in vitro transcription was obtained by PCR using the forward primer ccctaatacg actcactata gggaggaggg tttttaccat ggactgccat acaaaggag (SEQ ID NO: 1), which contains a T7 promoter (italicized), and a translation enhancer (bolded) (Al-Qahtani and Mensa-Wilmot, 1996, Nucleic Acids Res; 24:1173-1174). The first 21 nucleotides of the VSG—117 coding sequence are in regular style font. The reverse primer for VSG—11786 was cgaacaacga aggggttctt atagtgcgta gattcgtagc ttcgtttc (SEQ ID NO:2), and includes a HinfI site and nucleotides 78-86 of the coding region of VSG—117. One μl (1MBU) of RNase free DNase I was added to the reaction and allowed to incubate at 37° C. for 15 minutes (1 MBU digests 1 μg of pUC19 DNA in 10 minutes). An equal volume of TE-saturated phenol/chloroform was then added to the reaction, which was vortexed, and centrifuged (13,200 rpm, 15 minutes) at 4° C. The aqueous phase was withdrawn, combined with an equal volume of chloroform, vortexed and centrifuged (15 minutes, 13,200 rpm, 4° C.). To this aqueous phase 0.3M sodium acetate (final concentration) and precipitated with ethanol at −20° C. overnight. The precipitate was recovered by centrifugation (13,200 rpm, 4° C. for 15 minutes), rinsed with 70% ethanol, air-dried and dissolved 40 ml of nuclease free water. The RNA concentration was determined by measuring the absorbance at 260 nm, and confirmed by analysis with agarose gel electrophoresis and ethidium bromide staining.
Preparation of T. brucei microsomes (TbRM). Blood stream trypanosomes were obtained from the blood of infected rats, and purified by DE-52 chromatography. Cells (1×1010) were resuspended in 5 ml of homogenization buffer (made up of 250 mM Sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1 μg/ml TLCK, 5 μg/ml leupeptin, 0.5 mM PMSF (final concentrations). Two and half ml of resuspended cells were added to pre-chilled dounce homogenizer where they were lysed with 40 strokes of a pestle (specially selected for its tight-fitting to the homogenizer) on ice. After a one minute rest, another after 40 strokes was administered with the pestle. Homogenized cells were recovered and kept on ice while the remaining 2.5 ml of resuspended cells were being broken. Both sets of homogenized lysates were pooled and centrifuged (2,000 rpm, 10 minutes, 4° C.) in microfuge tubes. The supernatants were pooled, aliquoted into new microfuge tubes, and centrifuged at 12,000 rpm for 20 minutes at 4° C., and pellets resuspended in 40 μl (total volume) of fresh rough microsome buffer (RMB) (250 mM Sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM DTT, 0.5 μg/ml TLCK, 2.5 μg/ml leupeptin). The T. brucei microsome (TbRM) concentration was determined by measuring absorbance at 260 nm. Concentration of TbRM was adjusted (with RMB) to OD260 nm of 50, aliquoted, quick frozen in liquid nitrogen, and stored at −80° C. One equivalent of TbRM has an OD260 of 50). Supernatant from the 12,000 rpm centrifugation was also saved.
Preparation of cytosol from T. brucei. The supernatant obtained during the preparation of TbRM described above was centrifuged at 65,000×g (60 minutes, 4° C., Beckman TLA 100.3 rotor). Two milliliters of the resulting supernatant was concentrated twenty-fold by ultrafiltration with a Centricon-10 filter (Amicon). The retentate was retrieved, an aliquot was diluted 50-fold with 0.1% SDS, and the OD280 obtained. One equivalent of cytosol has an OD280 of 50. Aliquots were quick-frozen in liquid nitrogen, and stored at −80° C.
Protein import into TbRM. Two micrograms (μg) of RNA encoding a truncated substrate (e.g., VSG—11786) was translated in 40111 of a reaction mixture containing 20 μl rabbit reticulocyte lysate, 60 μM amino acid mixture (-Met, -Cys), and 2.4 μCi [35S]Promix. The reaction was incubated at 37° C. for 15 minutes. Cycloheximide (50 μg/ml, final concentration) was used to stop further translation, and the mixture was divided into two portions. T. brucei microsomes (ThRM) (1 equivalent) was added to one aliquot, and to the other portion an equal volume of RM buffer was added. The reactions were incubated at 37° C. for 45 minutes for protein import into TbRM to take place post-translationally.
Each reaction mixture was divided into three (each 10 μl) and treated with one of the following on ice for one hour: (I) RM buffer; (ii) 30 μg/ml protease K; (iii) nonidet P-40 (NP-40) (2%, final concentration) followed by protease K (30 μg/ml, final concentration). PMSF (20 mM final concentration) was added to stop protease K digestion. Samples were precipitated with an equal volume of cold (NH4)2SO4 sulphate (saturated) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4% total acrylamide, 3% cross linker in tricene-HCl system) (Fekkes et al., 1998, Mol Microbiol; 29(5):1179-90). The gels were dried, and radioactive polypeptides detected with a phosphorimager (Molecular Imager FX, (BioRad). Data bands were quantitated with QuantityOne software (BioRad), and graphs were plotted with DeltaGraph (Red Rock software).
Cell culture. Blood stream form T. brucei CA427 was cultured in HMI-9 medium (Hirumi and Hirumi, 1994, Parasitol Today; 10:80-84) to a maximum density of 106/ml. For use in drug sensitivity studies, cells were seeded at 10−4/ml, and one hundred microliters added to each well of a 96-well plate, with or without test compounds at stated concentrations, as discussed in Brief Descriptions of FIGS. 1-4. In controls, DMSO, the solvent for the compounds, was added. Cells were counted after 24 or 48 hours, using a hemocytometer, and the data graphed. All experiments were performed in triplicates.
HeLa cell line was maintained in RPMI-1640 supplemented with 10% bovine serum, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine at 37° C. in 5% CO2.
HeLa cell viability assays. Propidium iodide exclusion assay (reviewed in Steff et al., 2001, Cytometry; 45(4):237-43 and Wigg et al., 2003, Anal Biochem; 317(1):19-25) was used to determine HeLa cell viability after drug treatment. Cells were plated at 1×104 cells per well (96 well plate) with or without addition of test compounds for the stated time periods, as described for FIGS. 1-4. Etoposide stock (2 mM in DMSO) was and diluted in RPMI 1640 to 100 μM immediately before use. Adherent cells were rinsed with phosphate buffered saline (PBS), trypsinized (37° C. for 5 minutes), pelleted (2,000 g, 2 minutes), resuspended in PBS (500 μl) containing propidium iodide (1 μg/ml), DAPI [4′-6-Diamidino-2-phenylindole] (5 μg/ml) and analyzed by flow cytometry on a Dakocytomation CyAn without gating. Single fluorophore controls were acquired prior to the collection of the experimental data.
Mitochondrial XTT reduction assays (Ranta et al., 1998, J Cell Physiol; 176(1):92-8 and Berridge et al., 2005, Biotechnol Annu Rev; 11:127-52) were performed as follows. HeLa cells were seeded in 96 well plates (1×104 cells/well) (100 μl). At stated times, XTT dye (sodium 3,3-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro) (25 μl of 1 mg/ml), and 5-methylphenzanium sulfate (25 μM, final concentration) were added to each well. Plates were incubated for two hours (37° C., 5% CO2), and absorbance at 450 nm was read using a Versimax Tuneable Micro Plate Reader (Molecular Devices).
Results
T. brucei microsomes (TbRM) can import proteins post-translationally. Microsomes from canine pancreas are a model system used to study protein import into the ER of vertebrates and many other eukaryotes. However, ER signal sequences of T. brucei are not, in general, compatible with the protein import machinery of vertebrate ER (Al-Qahtani et al., 19998, Biochem J; 331:521-529 and Ramirez et al., 1999, J Eukaryot Microbiol; 46(6):557-65). Consequently, it was crucial, in order to study the biogenesis of trypanosome proteins such as VSG—11786, to develop a new protein import system using ER membranes from T. brucei. Two standard tests for successful microsomal ER protein import include proteinase (PK) protection of the translocated protein, and loss of protease protection after detergent permeabilization of microsomes (Connolly et al., 1989, J Cell Biol; 108(2):299-307). In the detergent permeabilization of microsomes test, detergent creates pores in the microsomal membranes that allow protease to access and digest imported proteins (Gelb et al., 2003, Mol Biochem Parasitol; 126(2):155-63).
A model trypanosome protein VSG—11786 was translated in rabbit reticulocyte lysate and cycloheximide was added to terminate protein synthesis. Subsequently, T. brucei microsomes (TbRM) was added, the mixture was incubated at 37° C. to facilitate protein import into TbRM, and reaction mixtures were (or were not) treated with proteinase K and/or detergent. See FIG. 1A for a flowchart of the experimental scheme. In the absence of TbRM, VSG—11786 was degraded by proteinase K (FIG. 1B, lane 2). TbRM protected VSG—11786 from proteinase K digestion (FIG. 1B, lane 4). Detergent permeabilization of TbRM resulted in proteinase K digestion of VSG—11786 (FIG. 1B, lane 5). From these data, it is concluded that VSG—11786 is imported post-translationally into TbRM. Signal peptide dependence of VSG—11786 translocation into TbRM was investigated. When the signal sequence was deleted from VSG—11786 the protein was not imported into TbRM.
From this example, it can be concluded that VSG—11786 is translocated into TbRM, a signal sequence is necessary for import of protein into TbRM, and import of VSG—11786 into TbRM occurs posttranslationally. Co-translational import of protein into TbRM could not be studied because the microsomes inhibit translation of protein.
A small molecule inhibitor of Hsp70-Hsp40 interaction blocks protein translocation into TbRM. Post-translational ER protein translocation in Saccharomyces cerevisiae and E. coli requires cytosolic chaperones. In the yeast S. cerevisiae, for example, Ssa1p (Hsp70) and co-chaperone Ydj1p (Hsp40) (Becker et al., 1996, Mol Cell Biol; 16(8):4378-86; Ngosuwan et al., 2003, J Biol Chem; 278(9):7034-42; and McClellan et al., 1998, Mol Biol Cell; 9(12): 3533-45) as well as Kar2p/BiP stimulate post-translational protein import (Lyman and Schekman, 1995, J Cell Biol; 131(5):1163-71; Panzner et al., 1995, Cell; 81(4):561-70; and Holkeri et al., 1998, J Cell Sci; 111:749-757). In E. coli, SecB and SecA chaperone protein secretion through the inner membrane (Zhou and Xu, 2003, Nat Struct Biol; 10(11):942-7; Ullers et al., 2004, Proc Natl Acad Sci USA; 101(20):7583-8; and Fekkes et al., 1998, Mol Microbiol; 29(5):1179-90), the topological equivalent of the eukaryote ER. Since protein import into T. brucei microsomes is post-translational the possibility that a trypanosome Hsp70 influenced protein translocation into TbRM was considered. Notably, the sequences of Ssa1p and a T. brucei cytosolic Hsp70 (Van der Ploeg et al., 1985, Science; 228(4706):1443-6) are 70% identical. To explore this concept, MAL3-101 was used as a small molecule inhibitor of Hsp40-mediated Hsp70 ATPase (Fewell et al., 2004, J Biol Chem; 279(49):51131-40) that inhibits import of prepro alpha-factor into microsomes from S. cerevisiae. T. brucei cytosol was pre-incubated with MAL3-101 (or an equal volume of DMSO), while VSG—117 mRNA was translated in a reticulocyte lysate. TbRM was then added post-translationally to the T. brucei cytosol-supplemented reticulocyte lysate, and after incubation, to promote import of protein into the microsomes, each mixture was treated with proteinase K. See FIG. 2A for flow chart of procedures.
In control reactions with DMSO in the reaction, TbRM imported 80% of VSG—11786, as measured by protection from proteinase K digestion (FIG. 2B, compare lanes 1, 2). In contrast, only 13% of VSG—11786 was translocated into TbRM when MAL3-101 was present (FIG. 2B, lanes 3, 4). In control experiments, MAL3-51 and MAL3-90 were examined. These data suggest that an Hsp70 family member is important for protein import into TbRM. More importantly, MAL3-101 is identified as a small molecule inhibitor of protein import into TbRM.
Equisetin and CJ-21,058 block protein translocation into TbRM. Post-translational protein secretion in E. coli is mediated by SecA (Watanabe and Blobel, 1993, Proc Natl Acd Sci USA; 90:9011-9015 and Knott and Robinson, 1994, J Biol Chem; 269(11):7843-6). CJ-21,058, isolated from the soil fungus CL47745, (Sugie et al., 2002, J Antibiot (Tokyo); 55(1):25-9) is reported to inhibit of SecA. Speculating that CJ-21,058 might inhibit post-translational import of protein into TbRM, the effect of the compound was tested on VSG—11786 import into TbRM, following a protocol outlined in the description of FIG. 1A. CJ-21,058 (20 μM) inhibited translocation of VSG—11786 into TbRM by 95% (FIG. 2B, compare lanes 7 and 8).
Equisetin, an analog of CJ-21,058, is produced by the soil fungus Fusarium heterosporum (Vesonder et al., 1979, J Antibiot (Tokyo); 32(7):759-61). Since CJ-21,058 blocked protein translocation into TbRM, it was tested if equisetin had similar effects. Equisetin (25 μM) inhibited translocation of VSG—11786 into TbRM by 95% (FIG. 2B, compare lanes 9 and 10). Hence, both CJ-21,058 and equisetin block VSG—11786 translocation into TbRM.
TbRM protein translocation blockers (PTBs) are trypanocidal. Many important cell surface proteins in T. brucei enter the secretory pathway via the ER (Steverding et al., 1995, J Cell Biol; 131:1173-1182). Consequently, it is anticipated that preventing the movement of proteins into the ER would reduce the amount of cell surface proteins some of which perform vital physiological functions, and lead to parasite death. Therefore, compounds that blocked protein translocation into TbRM (such as protein translocation blockers (PTBs)) were tested for trypanocidal activity against cultured trypanosomes.
MAL3-101 killed T. brucei in dose-dependent fashion (FIG. 3A). Fifty percent of parasites were killed at 125 nM (IC50), and one hundred percent of parasites were killed at 500 nM (IC100). The related compound MAL3-51 lacked trypanocidal activity at concentrations as high as 5 mM (FIG. 3B). Similarly, MAL3-90 did not kill T. brucei at 3 mM.
Equisetin and CJ-21,058 exhibited anti-trypanosomal properties. Equisetin had an IC50 of 3.3 μM (FIG. 3C). CJ-21,058 was also trypanocidal with an IC50 of 7 μM (FIG. 3D). In control studies using two assays of human HeLa cell viability, the amount of drugs that killed all trypanosomes had no measurable effect on host cells (FIG. 4).
It is concluded that inhibition of import of protein into TbRM is associated with trypanocidal action. Thus, the in vitro protein import system of the present invention will be valuable for identifying compounds that can kill T. brucei blood stream form.
To obtain a sense of the effects of the newly discovered anti-trypanosome compounds on host (vertebrate) cells, the drugs were tested on human HeLa cells using concentrations that killed one hundred percent T. brucei (IC100). No statistically significant differences were detected between drug-treated cells and control HeLa cells, using a cell viability assay based on propidium iodide exclusion (FIG. 4A). MAL3-101 (up to 10 mM) did not affect HeLa cell viability. Similarly, neither CJ-21,058 (20 μM) nor equisetin (26 μM) decreased viability of HeLa cells. In contrast, the positive control, etoposide, an inhibitor of topoisomerase II, produced significant cell death (FIG. 4A). Similar results were obtained with the XTT assay (FIG. 4B).
The data in FIGS. 1 to 4 are representative of many others performed that produced very similar results.
Discussion
Cell-free protein import into trypanosome ER membranes. An earlier study indicated that a signal peptide is important for secretion of a reporter protein in T. brucei (Bangs et al., 1996, J Biol Chem; 271:18387-18393). However, SRP is dispensable for protein entry into the secretory pathway (Liu et al., 2002, J Biol Chem; 277(49):47348-57), leading the authors of that study to propose the existence of an “alternative pathway” for protein entry into T. brucei ER. This example shows that T. brucei microsomes import proteins post-translationally (FIG. 1) in a signal sequence dependent manner. MAL3-101 an inhibitor of J-domain stimulated Hsp70 ATPase blocked import of VSG—11786 into TbRM (FIG. 2), consistent with the likely participation of a T. brucei homolog of an Hsp70 (cytosolic or localized to the ER) in ER protein import.
The properties of protein import into TbRM are reminiscent of observations made with fingi microsomes. S. cerevisiae and Candida maltosa microsomes can import proteins post-translationally (Hansen et al., 1986, Cell; 45:397-406; Brodsky et al., 1995, Proc Natl Acad Sci USA; 92(21):9643-6; Brodsky et al., 1993, J Cell Biol; 120:95-102; and Wiedmann et al., 1988, EMBO J; 7(6):1763-8), and a soluble factor that stimulated import of alpha-mating factor into S. cerevisiae microsomes (Waters et al., 1986, J Cell Biol; 103(6 Pt 2): p. 2629-36) was identified as Hsp70 Ssa1p) (Ngosuwan et al., 2003, J Biol Chem; 278(9):7034-42; McClellan et al., 1998, Mol Biol Cell; 9(12):3533-45; and Chirico et al., 1988, Nature; 332(6167):805-10), which interacts specifically with the J-domain protein Hsp40 (Ydj1p) (Becker et al., 1996, Mol Cell Biol; 16(8):4378-86; McClellan et al., 1998, Mol Biol Cell; 9(12): 3533-45; and Cyr and Douglas, 1994, J Biol Chem; 269(13):9798-804). These data suggest that T. brucei, like the yeasts, can use a chaperone-dependent pathway for protein translocation into the ER.
Trypanosome secretory pathway as a target for drug discovery. Cell surface proteins, for example VSG (Pays and Nolan, 1998, Mol. Biochem. Parasitol; 91:3-36 and Sheader et al., 2005, Proc Natl Acad Sci USA; 102(24): 8716-21), transferrin receptors (Steverding et al., 1995, J Cell Biol; 131:1173-1182), nucleobase transporters, and GP63 (El-Sayed and Donelson, 1997, J Biol Chem; 272:26742-26748) are important for establishment and maintenance of trypanosomatid infections in humans. Movement of these proteins to the plasma membrane is dependent on initial entry into the parasite ER, following paradigms worked out in model eukaryotes (reviewed in Wickner and Schekman, 2005, Science; 310(5753):1452-6). The ER is a gateway for targeting of proteins to the Golgi, endosomes, lysosomes and plasma membrane.
Due to the large number of proteins that transit through the ER as part of their biogenesis, it seems plausible the small molecules that interfere with import of proteins into the ER of parasites may have potential as anti-parasite agents. Such inhibitors are likely to have pleiotropic effects on parasite viability by inhibiting the biogenesis of cell surface receptors, nutrient transporters, and protective surface proteins.
In vertebrates, protein import into the ER is co-translational and dependent on signal recognition particle (SRP) (Rapoport et al., 1996, Annu Rev Biochem; 65:271-303). In contrast, several protein in T. brucei are imported into the ER without SRP (Liu et al., 2002, J Biol Chem; 277(49):47348-57). Further, T. brucei VSG—11786 and VSG MVAT7 can be imported into TbRM post-translationally (FIG. 1). Hence, both in vitro and in vivo data indicate that proteins can be imported into the ER of T. brucei post-translationally. In theory, mechanistic differences may exist between pathways used by vertebrate (hosts) and T. brucei to translocate proteins into the ER. If so, it is possible that compounds that interfere with post-translational ER protein import in T. brucei may compromise viability of the parasite without significant effect on vertebrate host cells, and become lead compounds for discovery of novel anti-trypanosome drugs. The complexity of the ER co-translational and post-translational protein translocation pathways (reviewed in Wickner and Schekman, 2005, Science; 310(5753): 1452-6) promises to offer multiple targets for drug discovery.
T. brucei ER protein translocation inhibitors (PTBs) are trypanocidal. Molecular chaperones (e.g., Hsp70, Kar2p(BiP), SecB, SecA) and co-chaperones (J-domain proteins Hsp40 and Sec63p) are important for post-translational protein import into the ER of eukaryotes, and for protein secretion in prokaryotes (for reviews see, Wickner and Schekman, 2005, Science; 310(5753):1452-6; Driessen et al., 2001, Nat Struct Biol; 8(6):492-8; and Mitra et al., 2006, Nat Struct Mol Biol; 13(11):957-64). The compound NSC 630668-R/1 (also called R/1) inhibits endogenous as well as Hsp40 stimulated Hsp70 activity (Fewell et al., 2004, J Biol Chem; 279(49):51131-40). Further, R/1 also inhibits post-translational protein translocation into ER microsomes from S. cerevesiae (Fewell et al., 2004, J Biol Chem; 279(49):51131-40 and Fewell et al., 2001, J Biol Chem; 276(2):910-4). Using combinatorial chemistry approaches, analogs of R/1 have been produced, and designated as MAL3 series (Fewell et al., 2004, J Biol Chem; 279(49):51131-40). MAL3-101 and MAL3-39 inhibit Hsp40 mediated ATPase of Hsp70, and MAL3-101 inhibits post-translational translocation of α-mating factor into yeast microsomes (Fewell et al., 2004, J Biol Chem; 279(49):51131-40).
This example studied the effect of small molecule modulators of Hsp70 on protein import into ThRM. MAL3-101 (structure shown in FIG. 5) blocked import of VSG—11786 into TbRM (FIG. 2, lanes 3 and 4). Surprisingly, MAL3-51 and MAL3-90 that inhibit intrinsic Hsp70 ATPase activity (Fewell et al., 2004, J Biol Chem; 279(49):51131-40) do not inhibit protein translocation into TbRM. The difference in results between MAL3-51 (or MAL3-90) and MAL3-101 suggests that J-domain protein (e.g., Hsp40 or Sec63) modulation of Hsp70 (or Kar2p) contributes to translocation of protein into TbRM. Thus, T. brucei homologs of Hsp70/Hsp40 or Kar2p/Sec63p may be important for protein import into ThRM.
MAL3 compounds were tested for their effect on cultured T. brucei. MAL3-101 killed T. brucei at sub-micromolar level (FIG. 4A) and had undetectable effect on a mammalian cell line (FIG. 4A). The IC50 of MAL3-101 is comparable to that of drugs currently to treat human African trypanosomiasis (HAT) (IC50 of suramin is 1.4-2.3 μM, pentamidine is 0.3 nM) (del Rayo Camacho et al., 2002, Phytother Res; 16(5):432-6). MAL3-51 and MAL3-90 were not trypanocidal, even at millimolar concentrations, consistent with their inability to inhibit protein translocation into TbRM. These data suggest that MAL3-101 is a lead compound for anti-trypanosome drug discovery.
Equisetin and CJ-21,058 are natural products from the fungii Fusarium heterosporum (Vesonder et al., 1979, J Antibiot (Tokyo); 32(7):759-61), and CL47745 (Sugie et al., 2002, J Antibiot (Tokyo); 55(1):25-9), respectively. They are acyl tetramic acid derivatives (pyrrolidine-2,4-diones) (Burke et al., 2000, Org Lett; 2(23):3611-3) some of which have antibiotic or antiviral activities. Equisetin may be produced in large amounts, since it has been synthesized chemically (Burke et al., 2000, Org Lett; 2(23):3611-3).
Both equisetin and CJ-21,058 inhibit import of proteins into TbRM (FIG. 2), and both compounds are trypanocidal (FIG. 3), with little effect on human HeLa cells (FIG. 4). IC50 of equisetin (3.3 μM) and CJ-21,058 (7 μM) is comparable to that of suramin that is used for treatment of HAT. Equisetin has been considered for treatment of HIV infection (reviewed in De Clercq, 2000, Med Res Rev; 20(5):323-49). Finally, the minimum concentration at which MAL3-101, equisetin, and CJ-21,058 exhibited trypanocidal effect (IC100) was used to test their protein translocation blocking effect on TbRM. All compound inhibited protein translocation into TbRM at the concentration used. This example demonstrates that inhibition of protein import into the ER is a mode of the anti-trypanosomal action of these compounds in vivo. Thus, MAL3-101, equisetin, and CJ-21,058 are lead compounds for anti-trypanosome drug development.
Example 3
Role of Cytosol in Translocation
The present invention has developed, for the first time, a cell-free microsomal system for import of trypanosome proteins into the parasite ER. This example shows that cytosol from T. brucei is required for translocation of the full-length variant surface glycoproteins VSG—117 and VSG_MVAT7 into T. brucei microsomes (TbRM).
Trypanosome microsome preparation. Following procedures also described in Examples 1 and 2, T. brucei blood stream form were harvested from infected rat blood, washed in phosphate buffered saline, and resuspended (2×109/ml) in homogenization buffer (50 mM HEPES-KOH, pH 7.5, 250 mM sucrose, 50 mM KOAc, 0.1 mM EDTA, 1 mM DTT). Each milliliter (ml) of parasites was lysed by Dounce homogenization, and cell lysis verified by microscopic examination. Lysates were centrifuged at 2,000-x g (10 minutes, 4° C.) to pellet nuclei and large aggregates. The supernatant from the first centrifugation was centrifuged at 12,000-x g (20 minutes, 4° C.), and the pellet saved as T. brucei rough microsomes (TbRM). The supernatant was centrifuged at 186,000-x g (4° C., 60 min) to obtain an 186,000 g-RM. All pellets were resuspended in rough microsome buffer (RMB) (at OD280=50 per ml) and stored at −80° C. until use. One microliter (μl) of this TbRM preparation equals one equivalent of microsomes.
Preparation of cytosol from T. brucei. To obtain cytosol, the supernatant from preparation of the 186,000 g-RM described above was filtered and concentrated. Two ml of supernatant was placed in an ultrafiltration tube (Microcon-10; molecular weight cut-off, 10 kDa) (Amicon). The tube was centrifuged (3,000-×g, 4° C., 120 min), producing a 40 μl retentate, the cytosol preparation that was frozen in liquid nitrogen, and stored at −80° C.
Coding sequences for “cargo proteins” (for ER translocation) were cloned downstream of a (phage) T7 promoter. mRNAs were synthesized in vitro with the Ampliscribe T7 transcription kit (Epicenter), and concentrated as directed by the kit manufacturer. Rabbit reticulocyte lysate (Promega) was used to synthesize potential cargo proteins (e.g., VSG—117). [35S]Methionine was added to the reticulocyte lysate, following vendor's instructions, in order to produce [35S]Met-labeled protein that could be monitored easily by phosphorimager detection.
Import of trypanosome proteins. As detailed in Example 2, rough microsomes from T. brucei (TbRM) import a truncated form of VSG—11786 (approximately 86 amino acids long (VSG86), termed mini-VSG)) synthesized in vitro. Protein import activity was associated with TbRM but not the 186,000 g-RM. Signal peptide dependence of mini-VSG translocation into TbRM was investigated. When the signal sequence was deleted from mini-VSG the protein was not imported. ER translocation of mini-VSG only works with T brucei microsomes, but not canine microsomes. A signal sequence is necessary for import of protein into TbRM, and translocation of mini-VSG into TbRM is posttranslational because TbRM was introduced into the reaction mixture after cycloheximide, which inhibits protein synthesis in T. brucei, had been added.
Cytosol from T. brucei is required for the import of a 27-kDa Protein into TbRM. Encouraged by the successful import of mini-VSG into TbRM, it was next attempted to import longer versions of the protein. A 114-amino acid long VSG—117 (VSG—117114) was imported efficiently but a substrate that was 260 amino acids long (VSG—117260) could not be translocated into TbRM. Similarly, full-length VSG—117 was not imported into TbRM under these circumstances. These results were baffling, because all substrates contained the same signal sequence that facilitated import of mini-VSG into TbRM.
Since all the protein translocation studied occurred posttranslationally, it was reasoned that a longer substrate might not translocate into TbRM because it may have adopted a conformation that was not “favorable” for import into the ER. In yeasts and E. coli that have posttranslational protein import systems, aggregated proteins can be “disentangled” by molecular “disaggregases” in the cytoplasm. Based on this information, it was suspected that cytosol from T. brucei might contain factors that could stimulate import of full-length VSG—117 into TbRM. To test this idea, cytosol was prepared from T. brucei, and used to supplement the in vitro system for translocation of VSG_MVAT7.
TbRM could not import full-length VSG_MVAT7 in the absence of cytosol. In the presence of cytosol, however, VSG_MVAT7 was protected from proteinase K. When detergent was added to permeabilize the microsomes prior to protease digestion, VSG_MVAT7 was degraded. Similar results were obtained with full-length VSG—117. Thus, it can be concluded that T. brucei cytosol contains a factor that is essential for posttranslational import of full-length VSG into TbRM.
Example 4
Small Molecule Inhibitors of In Vitro Protein Import onto the Trypanosome Endoplasmic Reticulum
Trypanosoma brucei causes human African trypanosomiasis (HAT), a disease against which new drugs are needed. To maintain an infection in vivo, T brucei relies on its cell surface proteins, including variant surface glycoproteins (VSGs). For VSG to be targeted to the exoplasmic side of the plasma membrane it must first translocate into the endoplasmic reticulum. Protein import into the ER is a good focus for the discovery of novel anti-trypanosome lead compounds. This example identifies compounds that inhibited protein translocation into ER microsomes, using an in vitro T. brucei microsomal system (TbRM) and a model VSG substrate (VSG-11786). Post-translational import of VSG-11786 into TbRM was inhibited by sodium azide, but not by valinomycin. Sodium azide is an inhibitor of F-type ATPases and SecA, thus an azide-sensitive chaperone is important for protein translocation into TbRM. The pyrmidinone-peptoid derivatives MAL3-101 and MAL3-51 inhibited post-translational import of VSG-11786 into microsomes. Compounds of this class modulate Hsp70 ATPase activity, and thus MAL3-101 and MAL3-51 likely affect ER protein import by inhibiting Hsp70 interaction with VSG-11786. Against cultured bloodstream T brucei, MAL3-101 was trypanocidal with an IG50 of 1.5 μM; however, MAL3-51 did not kill the parasite. A cyclopeptolide, NFI028, inhibited import of VSG-11786 into microsomes while a similar compound, CAM741, did not. Cyclopeptolides affect signal peptide recognition at the Sec61 translocon in a signal peptide-dependent manner. Thus, the signal peptide of VSG-11786 is inhibited at the Sec61 translocon by NFI028, but not by CAM741. Both NFI028 and CAM741 were trypanocidal with IG50's of 1.5 μM and 2.0 μM, respectively. Therefore, MAL3-101, NFI028, and CAM741 are lead compounds for the discovery of pyrimidinone-peptoid and cyclopeptolide derivatives as new anti-trypanosome drugs.
Central to T. brucei survival in a vertebrate is variant surface glycoprotein (VSG). Proper VSG expression on the exoplasmic leaflet of the plasma membrane is crucial for T. brucei viability (Cross, 1975, Parasitology; 71(3):393-417; Sheader et al., 2005, Proc Natl Acad Sci USA; 102(24):8716-21; reviewed by Taylor and Rudenko, 2006, Trends Genet; 22(11):614-20). VSGs are delivered to the plasma membrane by way of the secretory pathway. The committed step for protein entry into the secretory pathway is translocation into the endoplasmic reticulum (reviewed by Shimizu and Hendershot, 2007, Adv Exp Med Biol; 594:37-46); a process directed by ER signal peptides. ER signal peptides occur at the N-terminus of secretory precursor proteins (reviewed by Izard and Kendall, 1994, Mol Microbiol; 13(5):765-73; and Hegde and Bernstein, 2006, Trends Biochem Sci; 31(10):563-71), and must be recognized by a Sec61 translocon complex at the ER membrane (Simon and Blobel, 1991, Cell; 65(3):371-80; Crowley et al., 1994, Cell; 78(3):461-71; and Jungnickel and Rapoport, 1995, Cell; 82(2):261-70).
Signal peptides can be targeted to the ER by two pathways. Co-translational import is an ER import pathway that occurs as translation of a polypeptide on the ribosome is ongoing; it depends on a signal recognition particle (SRP) and an SRP-receptor (SR) at the ER membrane. In an alternative pathway, post-translational import, protein translocation into the ER occurs after full translation of the secretory precursor in the cytoplasm; it does not depend on SRP. In T. brucei, protein import into the ER occurs both co-translationally and post-translationally (Liu et al., 2002, J Biol Chem; 277(49):47348-57; Lustig et al., 2007, Eukaryot Cell; 6(10):1865-75; and Goldshmidt et al., 2008, J Biol Chem; 283(46):32085-98). This example evaluated the effects of small molecule compounds on post-translational import of VSG-11786 into T. brucei microsomes (TbRM).
Because Hsp70s in the cytoplasm and ER lumen are instrumental in protein translocation into the ER, compounds that inhibit their function could block VSG entry into the secretory pathway. Similarly, compounds that selectively inhibit T. brucei Sec61 translocon function could block VSG expression at the cell surface, and could be anti-trypanosome lead compounds.
The pyrimidinone-peptoid MAL3-101 (FIG. 6A) inhibits large T antigen (TAg, a J-domain containing protein) stimulation of Hsp70 ATPase activity, and inhibits post-translational translocation of pre-pro-alpha mating factor (pp MF) into S. cerevisiae microsomes (Fewell et al., 2004, J Biol Chem; 279(49):51131-40). With this example, the effects of MAL3-101 and a related compound, MAL3-51 (FIG. 6B) were tested on the post-translational import of VSG-11786 into TbRM.
Cyclopeptolides CAM741 (FIG. 6C) and NFI028 (FIG. 6D) inhibit co-translational translocation of certain secretory proteins in a signal peptide-dependent fashion. Both CAM741 and NFI028 inhibit secretion of alkaline phosphatase possessing a signal peptide from vascular cell adhesion molecule 1 (VCAM1) by interfering with signal peptide binding to the Sec61 translocon (Besemer et al., 2005, Nature; 436(7048):290-3; Harant et al., 2006, J Biol Chem; 281(41):30492-502; and Harant et al., 2007, Mol Pharmacol; 71(6):1657-65). However, CAM741, but not NFI028, inhibited protein import directed by a vascular endothelial growth factor (VEGF) signal peptide. Because both CAM741 and NFI028 inhibit ER protein import with non-identical selectivity of signal peptides, both compounds were tested on VSG-11786 import into TbRM.
Azide interferes with a variety of cellular processes across biological kingdoms. Growth of bloodstream T. brucei is inhibited by azide (IG50 of 100 μM); however, it is not known how azide affects T. brucei (Steverding and Scory, 2004, J Parasitol; 90(5):1188-90). In E. coli and other prokaryotes, azide blocks protein translocation in vivo and in vitro by inhibiting the ATPase of SecA, a protein translocation chaperone (Oliver et al., 1990, Proc Natl/Acad Sci USA; 87(21):8227-31; van der Wolk et al., 1997, EMBO J; 16(24):7297-304; and reviewed by Schmidt and Kiser, 1999, Microbes Infect; 1(12):993-1004). Based on data showing T. brucei azide sensitivity and the ability of azide to inhibit post-translational protein translocation in prokaryotes, whether sodium azide affected post-translational translocation of VSG-11786 into TbRM was tested.
This example identifies pyrimidinone-peptoids and cyclopeptolides as two classes of compounds that inhibit T. brucei ER protein import. Further, some of the compounds are trypanocidal. Therefore, pyrimidinone-peptoids and cyclopeptolides are lead compounds for discovery of new anti-trypanosomal compounds.
Materials and Methods
Materials. Taq DNA polymerase, amino acid mixture minus methionine and cysteine, and nuclease treated rabbit reticulocyte lysate were obtained from Promega (Madison, Wis.). dNTPs were obtained from Sigma (St. Louis, Mo.). DEPC-treated water was from USB Corporation (Cleveland, Ohio); QIAquick PCR purification kit was from Qiagen (Valencia, Calif.); Ampliscribe T7 in vitro transcription kit was from Epicentre (Madison, Wis.); TRAN35S—LABELTM No-thaw metabolic labeling reagent was from MP Biomedicals (Solon, Ohio); cycloheximide was from Calbiochem (La Jolla, Calif.); proteinase K and leupeptin were from Roche (Indianapolis, Ind.); PMSF was from Boehringer Mannheim (Mannheim, Germany); ethanol and urea were from Fisher Scientific (Norcross, Ga.); Acrylagel and Bis-Acrylagel were from National Diagnostics (Atlanta, Ga.); TEMED and ammonium persulfate, were from Bio-Rad (Hercules, Calif.); DE52 was from Whatman (Hillsboro, Ohio); MAL3-101 and MAL3-51 were provided by Dr. Jeffrey L. Brodsky and Dr. Peter Wipf (University of Pittsburgh); AEE788, CAM741 and NFI028 were provided by Novartis (Vienna, Austria); sodium azide was from J. T. Baker (Phillipsburg, N.J.); valinomycin and all other chemicals and reagents were obtained from Sigma (St. Louis, Mo.).
Plasmids. VSG-11786 DNA was made through PCR mutagenesis of a plasmid template, pVSG-117 (Innis et al., 1988, Proc Natl Acad Sci USA; 85(24):9436-40; Bangs et al., 1996, J Biol Chem; 271:18387-93).
Cell Strain and Isolation. Monomorphic Trypanosoma brucei strain 427 were grown in and purified from Sprague Dawley rats, using chromatography on DE52 (Cross 1975).
Primer Construction For VSG-11786. PCR mutagenesis was used to create VSG-11786 from the template pVSG-117 using variations of the forward primer TAATACGACT CACTATAGGG aggagggttt ttaccATGGA CTGCCATACA AAGGAG (SEQ ID NO:22). Each primer contained a T7 promoter (upper case) and a translational enhancer (lower case) (Teilhet et al., 1998, Gene; 222(1):91-7). The coding region of VSG-117 starts at position 36 and ends at primer position 56 (upper case). The VSG-117 protein was truncated to 86 amino acids, using the reverse primer cgaacaacga aggggttcTT ATAGTGCGTA GATCGTAGCT TCGTTTC (SEQ ID NO:23), which has a stop codon (upper case) after the 27 nucleotides priming for nucleotides 295-321 (upper case) of the pVSG-117 sequence.
Generation of VSG-11786 DNA. A VSG-11786 PCR product was generated in a 100 μl reaction mixture containing 224 ng pVSG-117 template, 0.5 μM of each forward and reverse primer, Innis buffer (10 mM Tris-HCl pH 8, 2.5 μM MgCl2, 0.05% Tween-20, 0.05% nonidet P-40, 50 mM KCl) (Innis et al., 1988, Proc Natl Acad Sci USA; 85(24):9436-40), 250 μM dNTPs, and 5 units of Taq DNA polymerase. The PCR reaction was carried out for 25 cycles as follows: 95° C. for 90 seconds, 56° C. for 90 seconds, and 74° C. for 2 minutes. The PCR product was purified using a QIAquick PCR purification kit (Qiagen). Purified PCR products were quantitated at 260 nm.
In Vitro Transcription. One μg of purified DNA was used as a template for transcription with an AmpliscribeTM T7 kit (Epicentre Technologies). One μl (1 MBU) of RNase free DNase I was added to the reaction (20 μl) and allowed to incubate at 37° C. for 15 minutes. The mixture was extracted with an equal volume of TE-saturated phenol/chloroform, and the aqueous phase extracted with chloroform. To the aqueous phase, sodium acetate (27 mM final concentration) was added along with 2.5 times the total volume of 100% ethanol. This mixture was allowed to precipitate at −20° C. overnight. The precipitate was recovered at 16,100×g at 4° C. for 15 minutes, rinsed with 70% ethanol, and pelleted at 16,100×g. The pellet was allowed to air dry, and resuspended in 40 μl of nuclease free water. The RNA concentration was determined by measuring the absorbance at 260 μm.
Microsome Preparation from Bloodstream Trypanosoma brucei. Bloodstream T. brucei lister 427 (1×1010 cells/ml) were resuspended in 5 ml of fresh homogenization buffer (HB) (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1 μg/ml TLCK, 5 μg/ml leupeptin, 0.5 mM PMSF). In a clean, pre-chilled dounce homogenizer 2.5 ml of the resuspended cells were lysed on ice by 2 repetitions of 40 strokes with a tight-fitting pestle, with a one minute break after 40 strokes. The homogenates were pooled, aliquoted into microcentrifuge tubes (1 ml/tube), and centrifuged at 400×g for 10 minutes at 4° C. The supernatants from all tubes were pooled, aliquoted into a new microcentrifuge tube, and centrifuged at 13,400×g for 20 minutes at 4° C. The supernatants from the 13,400×g centrifugation step were recovered and pooled. Pellets from the 13,400×g centrifugation step were resuspended in 50 μl/pellet (total volume) of fresh rough microsome buffer (RMB) (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 1 mM DTT, 0.5 μg/ml TLCK, 2.5 μg/ml leupeptin). The microsome concentration was determined by measuring the absorbance at 260 nm, and the concentration was adjusted to an OD260 of 50 (1 equivalent with an OD260 of 50 μl). The microsomes were aliquoted into 20 μl portions, quick frozen in liquid nitrogen, and stored at −80° C. (Pellets from the 400×g centrifugation and the supernatant from the 13,400×g centrifugation were also saved).
Cytosol Preparation from Bloodstream Trypanosoma brucei. The supernatant saved from the 13,400×g centrifugation step (above) during the microsome preparation was centrifuged at 179,500×g at 4° C. for one hour in a Beckman Coulter Optima TLX ultracentrifuge. The supernatant was recovered, pooled (approximately 4 ml total), and concentrated using a Centricon-10 filter by centrifuging at 5000×g at 4° C. in 30 minute intervals until approximately 250 μl of concentrated cytosol remained. The cytosol was adjusted to an OD280 of 50 (1 equivalent=50 OD units/μl), aliquoted, quick-frozen in liquid nitrogen, and stored at −80° C.
In Vitro Translation and Import of VSG Into T. brucei Microsomes (TbRM). RNA (250-500 ng) in DEPC-treated H2O (7 μl) was incubated at 65° C. for three minutes. After cooling on ice for one minute, 1.5 μl (75 μM final concentration) of an amino acid mixture lacking methionine and cysteine (Promega), 1.5 μl of 35S-cysteine and methionine (2.5 mCi total activity, MP Biomedicals), 10 μl of rabbit reticulocyte lysate (Promega), and 1 μl of T. brucei cytosol was added to the RNA mixture, bringing the final volume to 21 μl. This reaction mixture was incubated at 37° C. for one hour. Translation was stopped with cycloheximide (1 mM final concentration).
The translation reaction was then divided into four separate portions of 5 μl each. Two of these portions were left untreated while the remaining two portions were supplemented with 1 μl (1 μl=1 equivalent, which has an OD260 of 50) of TbRM. For inhibitor studies, one of the following was then added: MAL3-101 (10 μM final concentration), MAL3-51 (10 μM final concentration), CAM741 (1 μM final concentration), NFI028 (1 μM final concentration), sodium azide (8 μM final concentration), or AEE788 (8 μM final concentration). The four reaction aliquots were further incubated at 37° C. for 1.5 hours and then transferred to ice. Next, one of each sample (translation mixture with or without TbRM) was treated with a proteolysis mixture containing urea (3M) and proteinase K (0.5 mg/ml) (final concentrations) on ice for one hour. PMSF (34 mM final concentration) was added to quench the proteolysis reaction. Proteins were precipitated with 60% (NH4)2SO4 on ice for 15 minutes, and centrifuged at 16,100×g at 4° C. for 7 minutes. The pellets were resuspended in 20 μl of 2.5×SDS sample buffer and resolved by SDS-PAGE in a Tris-Tricine gel system (16%—Resolving; 3%—Stacking) (reviewed by Gallagher, 2007 “One-dimensional SDS gel electrophoresis of proteins,” Curr Protoc Cell Biol Chapter 6: Unit 6.1)). Radioactive polypeptides were detected with a phosphorimager; bands were quantitated with Quantity-One software (version 4.6.5, Bio-Rad).
Quantitation of Percent VSG Import. Using Quantity-One software (version 4.6.5, Bio-Rad), phosphorimages were adjusted so that gel bands corresponding to proteinase K-protected VSG-11786 were visible. Next, background noise signals were subtracted using the “filter wizard” application. A Tiff image preserving these adjustments to the gel image was acquired. From this Tiff image, gel bands in each lane were quantitated by measuring the volume (counts*mm2) of the band representing VSG-11786. For background signal, the volume of an area in each lane of the gel image that best represented the average background pixel intensity was measured. In band and background intensity quantitation, the dimensions of the volume boxes were maintained. Next, a volume analysis report was performed to calculate the “adjusted volumes” (volume of VSG-11786 band−volume of average background bands) of the VSG-11786 bands. VSG import percentages were obtained as follows: the “adjusted volume” corresponding to a band detected in the presence of TbRM and challenged with proteinase K was divided by the “adjusted volume” corresponding to a band detected in the presence of TbRM without proteinase K. This quotient was multiplied by 100 to obtain a percentage of VSG import.
Cell Culture. Bloodstream T. brucei cell culture and drug susceptibility studies were performed as previously described. Bloodstream T. brucei CA427 were cultured in HMI-9 media (Hirumi and Hirumi, 1994, Parasitol Today; 10(2):80-4) to a density of 106 cells/ml. Cells were seeded (500 μl of 4×104 cells/ml) to a 24-well plate and exposed to MAL3-101, CAM741, and NFI028 at listed concentrations (see figure legends) or an equal volume of DMSO. Cells were incubated at 37° C. and counted after 24 and 48 hours, using a hemocytometer.
Results
MAL3-101 and MAL3-51 Inhibit Post-Translational Import of VSG-117 into T. brucei Microsomes. Cytosolic chaperones are essential for post-translational protein import into the ER (McClellan et al., 1998, Mol Biol Cell; 9(12):3533-45; McClellan and Brodsky, 2000, Genetics; 156(2):501-12; Ngosuwan et al., 2003, J Biol Chem; 278(9):7034-42; and Wickner and Schekinan, 2005, Science; 310(5753):1452-6). Specifically, homologues of Hsp70 and its co-chaperone Hsp40 facilitate import in yeast by rendering secretory precursors “import competent” (Caplan et al., 1992, Cell; 71(7):1143-55; McClellan and Brodsky, 2000, Genetics; 156(2):501-12; Ngosuwan et al., 2003, J Biol Chem; 278(9):7034-42). MAL3-101 inhibits Hsp40 stimulation of Hsp70 ATPase, and inhibits post-translational translocation of pp MF into yeast microsomes (Fewell et al., 2004, J Biol Chem; 279(49):51131-40). In T. brucei, VSG can enter ER microsomes (TbRM) post-translationally. Since post-translational VSG translocation into ThRM may depend on cytosolic chaperones, the effects of MAL3-101 and MAL3-51 on import of the protein into TbRM were tested. As a model substrate we used VSG-117 that is truncated to 86 amino acids because unlike longer substrates it appears to have less stringent requirements for efficient import into TbRM.
MAL3-101 (10 μM) reduced VSG-11786 import into ThRM by 54% compared to a DMSO control (FIGS. 7A and 7B). Thus, MAL3-101 inhibits post-translational import of VSG-11786 into TbRM. In T. brucei, cytosol is crucial for post-translational import of full-length VSG into ThRM. Therefore, the effect of T. brucei cytosol on VSG-11786 import into TbRM in the presence of MAL3-101 was tested. T. brucei cytosol suppressed MAL3-101 inhibition of VSG-11786 import into TbRM; 90% of VSG-11786 was imported into TbRM in the presence of MAL3-101 (10 μM) (FIGS. 7A and 7B).
MAL3-51 (10 μM) also reduced import of VSG-11786 into TbRM by 41% compared to the control (FIGS. 7A and 7B). However, T. brucei cytosol failed to rescue the MAL3-51 inhibition of VSG-11786 import into TbRM; 45% of VSG-11786 was imported into TbRM in the presence of MAL3-51 (10 μM) (FIGS. 7A and 7B).
This data demonstrates that the target of MAL3-101 is most likely a cytosolic protein (or factor); excess quantities of it from T. brucei cytosol bypass inhibition by the compound. On the other hand, excess amounts of the target of MAL3-51 are not present in cytosol from T. brucei. 
The effects of MAL3-101 and MAL3-51 on VSG import into TbRM suggested that these compounds might inhibit translocation of other proteins in vivo, and compromise viability of T. brucei. Therefore, whether MAL3-101 and MAL3-51 were trypanocidal was tested. MAL3-101 inhibited cell growth (IG50 of 1.5 μM), killing all parasites at 3 μM (FIG. 7C). However, MAL3-51 did not kill T. brucei at concentrations up to 500 μM.
NFI028 Inhibits VSG-11786 Import into TbRM. CAM741 and NFI028 (FIGS. 6C and 6D) are cyclopeptolides, both of which inhibit mammalian co-translational protein import into the ER in a signal peptide-dependent fashion (Besemer et al., 2005, Nature; 436(7048):290-3; Harant et al., 2006, J Biol Chem; 281(41):30492-502; Harant et al., 2007, Mol Pharmacol; 71(6):1657-65). The effects of NFI028 and CAM741 on post-translational import have not been tested. In general however, T. brucei signal peptides are different from most mammalian signal peptides and are not translocated into canine microsomes (Al-Qahtani et al., 1998, Biochem J; 331:521-9). Based on these facts, the effects of NFI028 and CAM741 on import of VSG-11786 into TbRM was tested to evaluate their possible effects on T. brucei VSG-117.
NFI028 (1 μM) inhibited VSG-11786 translocation into TbRM by 49%, compared to a DMSO control (FIGS. 8A and 8B). Higher concentrations of NFI028 (5 and 10 μM) inhibited VSG-11786 import into TbRM to the same degree. In contrast, CAM741 (1 μM) did not affect import; VSG-11786 import into TbRM was 73% and 74% in the presence of CAM741 or DMSO (solvent), respectively (FIGS. 8A and 8B). Similar results were obtained when import of VSG-11786 into TbRM was tested with 10 μM CAM741.
NFI028 and CAM741 are Trypanocidal. Based on the observation that NFI028 inhibited post-translational import of VSG-11786 into TbRM, it was determined if NFI028 was trypanocidal. If NFI028 inhibited ER protein import of VSG-11786 in the present cell-free system it might also block translocation of other proteins in vivo. CAM741 was studied as a control compound. Upon exposure of bloodstream T. brucei to NFI028, the cells were killed (IG50 of 1.5 μM) (FIG. 8C). Surprisingly, cells exposed to CAM741 were also killed with an IG50 of 2 μM (FIG. 8D).
Sodium Azide Inhibits Post-Translational Import of VSG-11786 into TbRM. Azide is a metabolic poison (Detimary et al., 1994, Biochem J; 297 (Pt 3):455-61; Van de Casteele et al., 2003, Biochem Biophys Res Commun; 312(4):937-44; Bowler et al., 2006, Proc Natl Acad Sci USA; 103(23):8646-9) that inhibits growth of bloodstream T. brucei (Steverding and Scory, 2004, J Parasitol; 90(5):1188-90). Azide inhibits F-type ATPases (Bowler et al., 2006, Proc Natl Acad Sci USA; 103(23):8646-9) and SecA, blocking protein secretion in prokaryotes (Oliver et al., 1990, Proc Natl Acad Sci USA; 87(21):8227-31; van der Wolk et al., 1997, EMBO J; 16(24):7297-304; Miller et al., 2002, Biochemistry; 41(16):5325-32). To test the nature of (chaperone) ATPases that might influence post-translational translocation of VSG-11786 into TbRM, the effects of azide and AEE788 were tested. AEE788 is an ATP mimic which inhibits kinase activities of endothelial growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) (Traxler et al., 2004, Cancer Res; 64(14):4931-41; Park et al., 2005, Clin Cancer Res; 11(5):1963-73; and Yazici et al., 2005, Prostate; 65(3):203-15).
When VSG-11786 import into TbRM was tested in the presence of sodium azide (8 μM), post-translational import of the protein into TbRM was inhibited by 54% (FIGS. 9A and 9B). Similarly, VSG-11786 import into TbRM was inhibited by 20% with 4 μM sodium azide. However, AEE788 (8 μM) failed to inhibit post-translational import of VSG-11786 into TbRM (FIGS. 9A and 9B).
Next, it was determined if sodium azide was inhibiting ER protein import by acting on a membrane component of TbRM. This was accomplished by testing import of VSG-11786 into TbRM that had been pre-treated with sodium azide (8 μM); TbRM pre-treated with NFI028 (1 μM) was used as a control. TbRM were treated with concentrations of sodium azide or NFI028 that were previously shown to inhibit ER protein import (FIGS. 9A-B and 8A-B, respectively). Addition of pre-treated microsomes to the reaction mixture reduced the effective concentrations of sodium azide and NFI028 from 8 μM and 1 μM to 1.4 μM and 0.2 μM, respectively. Protein import of VSG-11786 into azide-treated microsomes was not inhibited (FIGS. 9C-D), suggesting that azide does not act on TbRM membranes. As a control, TbRM were pre-treated with NFI028 (1 μM) and tested for protein import. Import of VSG-11786 into these NFI028-treated microsomes was inhibited by 10% (FIGS. 9C-D), indicating that, unlike sodium azide, NFI028 could act on TbRM membranes, or NFI028 inhibits protein import into TbRM when present at low concentrations in the reaction mixture.
Azide inhibits cytochrome c oxidase leading to a reduction of mitochondrial membrane potential, and blocks protein translocation into mitochondria (Bennett et al., 1992, J Geriatr Psychiatry Neurol; 5(2):93-101). However, bloodstream form T. brucei do not possess a functional mitochondria and lack cytochromes (Vickerman, 1985, Br Med Bull; 41(2):105-14; Gull, 2002, Curr Pharm Des; 8(4):241-56; Steverding and Scory, 2004, J Parasitol; 90(5):1188-90). Therefore, azide is unlikely to reduce mitochondrial membrane potential in bloodstream T. brucei. Nevertheless, to ensure that the inhibitory effect of azide on VSG-11786 import was occurring at ER-derived TbRM and not indirectly through inhibition of mitochondrial functions, the effect of valinomycin on VSG-11786 post-translational import was tested. Valinomycin is a potassium ionophore that lowers membrane potential and inhibits mitochondrial protein import (at 0.5 μM) in T. brucei (Yermovsky-Kammerer and Hajduk, 1999, Mol Cell Biol; 19(9):6253-9; Priest and Hajduk, 2003, J Biol Chem; 278(17):15084-94). The efficiency of VSG-11786 import into TbRM was not affected by 0.5 μM valinomycin (FIGS. 9C-D), a concentration shown to inhibit mitochondrial protein import (Priest and Hajduk, 2003, J Biol Chem; 278(17):15084-94).
This data demonstrate that sodium azide reduces post-translational import of VSG-11786 into TbRM. However, azide does not have a direct effect on the T. brucei ER membrane.
Discussion
MAL3-101 and MAL3-51 Inhibit TbRM Protein Import But Only MAL3-101 Kills T. brucei. MAL3-101 inhibits Hsp40-stimulated Hsp70 activity (Fewell et al., 2004, J Biol Chem; 279(49):51131-40). In T. brucei, MAL3-101 inhibited post-translational VSG-11786 import into TbRM (FIGS. 7A-B). However, cytosol from the parasite suppressed MAL3-101 inhibition of protein import (FIGS. 7A-B). T. brucei cytosol contains proteins that could function as chaperones necessary for post-translational protein import. Cytosol may reduce MAL3-101 inhibition for a variety of reasons: one possibility is that the addition of cytosol raised the concentration of Hsp40, excess amounts of which overcame the effect of the inhibitor. There is precedent for this phenomenon: MAL3-101 inhibition of T-antigen (TAg) stimulated Hsp70 ATPase activity could be suppressed with excess amounts of TAg (Fewell et al., 2004, J Biol Chem; 279(49):51131-40).
MAL3-51 inhibited import of VSG-11786 into TbRM in the absence of T. brucei cytosol, but unlike MAL3-101, cytosol did not block MAL3-51 effects on VSG-11786 import into TbRM (FIGS. 7A-B), indicating that MAL3-51 and MAL3-101 inhibit post-translational protein import into TbRM by different mechanisms. First, MAL3-51 could inhibit Hsp70 chaperoning of VSG-11786, but not by inhibiting Hsp40/Hsp70 ATPase stimulation. Second, MAL3-51 could interact with different types of Hsps than those bound by MAL3-101. As an example of such phenomenon, MAL3-101 inhibits TAg stimulation of Hsp70 ATPase, but does not inhibit Ydj1 (Hsp40) stimulation of Hsp70 ATPase activity (Fewell et al., 2004, J Biol Chem; 279(49):51131-40).
MAL3-101 is trypanocidal, inhibiting bloodstream T. brucei growth in vitro (IG50 of 1.5 μM) (FIG. 7C). This data indicate that MAL3-101 can cross the plasma membrane of the parasite to exert its effects. Given the diverse roles of Hsp40/Hsp70 chaperones in vivo, this example indicates that MAL3-101 affects ER protein import as well as a variety of other cellular processes (such as, protein folding, protein import into the nucleus, protein import into glycosomes, signal transduction pathways) (reviewed in Brodsky and Chiosis, 2006, Curr Top Med Chem; 6(11):1215-25).
MAL3-51 did not kill bloodstream T. brucei cultured in vitro. When the structures of MAL3-101 and MAL3-51 are compared (FIGS. 6A-B), it is evident that the difference in abilities of MAL3-101 and MAL3-51 to inhibit protein import into TbRM and kill T. brucei may be a direct result of their structural differences. MAL3-101 possesses a pyrimidinone linked to a peptoid group by a hexylamino group (FIG. 6A, grey boxes) (Wright et al., 2008, Bioorg Med Chem; 16(6):3291-301). MAL3-51 has a peptoid group, but does not possess a pyrimidinone group (FIG. 6B) (Wright et al., 2008, Bioorg Med Chem; 16(6):3291-301). Also, the peptoid scaffolds of MAL3-101 and MAL3-51 have different substituents. Studies in mammalian tumor cells suggest that substituents off of the pyrimidinone group (R1) and peptoid group (R3) (FIG. 6A) are crucial for MAL3-10's ability to inhibit tumor growth (Wright et al., 2008, Bioorg Med Chem; 16(6):3291-301). MAL3-51 does not have a pyrimidinone group as seen in MAL3-101, and thus lacks a chemical group at the R1 position (compare FIGS. 6A and 6B). Ultimately, these chemical differences may affect their cell permeability properties, causing MAL3-101 to be more effective than MAL3-51 in vivo (Wright et al., 2008, Bioorg Med Chem; 16(6):3291-301).
Two Related Cyclopeptolides have Different Effects on VSG Import into TbRM. NFI028, but not CAM741, inhibited VSG-11786 import into TbRM (FIGS. 8A-B). This example is the first demonstration that NFI028 inhibits signal peptide function in a post-translational system. In a mammalian system, CAM741 and NFI028 inhibit ER protein import of a select group of signal peptides (Besemer et al., 2005, Nature; 436(7048):290-3; Harant et al., 2006, J Biol Chem; 281(41):30492-502; Harant et al., 2007, Mol Pharmacol; 71(6):1657-65). Specifically, CAM741 inhibits ER import of proteins whose signal peptides are relatively inefficient at directing import, and those with “helix-breaking” residues (glycine and proline) in their signal peptides. It is suggested that CAM741 prevents proper association of the signal peptide with the Sec61 translocon by binding directly to the signal peptide and/or by competitively associating with the signal peptide-binding site at the translocon (Harant et al., 2007, Mol Pharmacol; 71(6):1657-65).
Both NFI028 and CAM741 inhibited T. brucei growth, with similar IG50 concentrations (1.5 μM and 2.0 μM, respectively) (FIGS. 8C-D). This data suggests that CAM741, along with NFI028, could inhibit other signal peptides in vivo thereby compromising viability of T. brucei. Also, the data indicates that CAM741 and NFI028 are transported into T. brucei, and thus are candidates for anti-trypanosome lead compounds. Furthermore, NFI028 and CAM741 have differences in their structures (FIGS. 6C-D, light grey box); NFI028 possesses a piperidine-1-carbaldehyde substitution while CAM741 is substituted with a propyl propionate group in that position (FIGS. 6C-D, dark grey box). These differences in structure produce unique effects on the VSG-117 signal peptide. CAM741 could inhibit other signal peptides in vivo, but not VSG-117. This indicates that derivatives of cyclopeptolides may posses anti-trypanosomal properties.
Azide Inhibits Post-Translational Import of VSG-11786 Into TbRM. Post-translational import of VSG-11786 into TbRM was inhibited by sodium azide (FIGS. 9A-B), but protein translocation was not inhibited when TbRM was pre-incubated with sodium azide (FIGS. 9C-D). AEE788, a competitive inhibitor of ATP for protein tyrosine kinases, had no effect on protein translocation (FIGS. 9A-B). These data indicated that azide did not inhibit all ATP-binding proteins, but blocked a select group of ATP-binding proteins. Further, valinomycin, an inhibitor of mitochondrial protein import, did not alter VSG-11786 import into TbRM (FIGS. 9C-D) at a concentration shown to inhibit mitochondrial protein import, verifying that azide inhibited protein import at ER-derived membranes and not some mitochondrial contaminant of ThRM. These data demonstrate that azide inhibits a cytosolic chaperone that is important for VSG import into TbRM. Azide inhibits F-ATPases and SecA ATPase activity by forming a tight complex with bound-ADP in the ATPase catalytic site (Bowler et al., 2006, Proc Natl Acad Sci USA; 103(23):8646-9), preventing the exchange of ADP for ATP. Since ATP hydrolysis is needed to drive SecA-dependent protein translocation, prokaryotic protein secretion is blocked in the presence of azide because the site of ATP hydrolysis is competitively inhibited by the ADP-azide complex (Oliver et al., 1990, Proc Natl Acad Sci USA; 87(21):8227-31; van der Wolk et al., 1997, EMBO J; 16(24):7297-304; Miller et al., 2002, Biochemistry; 41(16):5325-32; Bowler et al., 2006, Proc Natl Acad Sci USA; 103(23):8646-9). One possible explanation for azide sensitive ER protein import observed in T. brucei is that T. brucei could possess an azide-sensitive SecA-like cytosolic chaperone that is crucial for post-translational protein translocation into the ER. Alternatively, azide might inhibit chaperones with F-ATPase domains, halting protein translocation through the Sec61 channel in TbRM.
Protein translocation chaperones (e.g. Hsp40/Hsp70) and ER signal peptides are essential to post-translational protein import into the ER. Using a T brucei in vitro translocation system, this example has shown that protein translocation into TbRM can be blocked by small molecule inhibitors that target these components. The properties of these inhibitors will be further explored for the discovery of new anti-trypanosome lead compounds.
Example 5
Specificity of TbRM for Signal Peptides from Different Biological Families
Several studies have indicated that trypanosomatid signal peptides are not compatible with the translocon in vertebrates. However, T. brucei microsomes (TbRM) import proteins from the parasite, as expected. This example determined that the species-specificity of signal peptide utilization of canine microsomes is also exhibited by T. brucei microsomes. For this purpose, proteins from E. coli (β-lactamase), S. cerevisiae (prepro-alpha mating factor), Bos taurus (bovine) (preprolactin) and Leishmania major (gp63) were each translated in vitro and incubated with microsomes and cytosol from T. brucei. Translocation of the proteins into ThRM was tested with protease protection assays, as described in the previous examples.
Trypanosome TbRM could not protect proteins from the other species listed above from digestion by protease K. In control experiments, VSG—117 was protected from the protease under identical conditions. From these data, it can be concluded the TbRM is specific, in general, for signal peptides from T brucei. Failure of gp63 to enter TbRM was surprising since L. major is a trypanosomatid. However, the composition and design of Leishmania major signal peptides is similar to those from Gram-positive bacteria whereas T. brucei signal sequences are not very similar in composition or organization of the subdomains to the prokaryote signal sequences.
Example 6
Defining Properties of Trypanosoma brucei Signal Peptides
Plasma membrane proteins are important for cellular communication and viability. Movement of proteins to the plasma membrane requires translocation into the endoplasmic reticulum (ER). An N-terminal signal sequence is required for import of proteins into the ER. In T. brucei, rudimentary properties of the ER protein translocation system have not been studied, despite the glaring importance of the pathway in the parasite. For instance, the essential features of signal peptides have not been defined. Although signal sequences had been thought to be interchangeable between species, in general, signal peptides from T. brucei are not functional with canine microsomes that are widely used to study mechanisms of ER protein import. ER signal peptides contain an h-region that is thought to be composed of random hydrophobic amino acids. Bioinformatic analyses reveal that h-regions of T. brucei contain conserved peptide patterns (motifs). Human h-regions contain motifs that are different from those in T. brucei. 
The objective of this example is to provide a comprehensive account of the pathways and mechanisms of protein import into the ER of trypanosomes. Accomplishing this goal requires a study of both signal peptides and factors that are required for translocation of protein into TbRM. By comparing the properties of the trypanosome protein import pathway with those of mammalian host cells, new lead anti-trypanosome compounds will be identified.
In this example, T. brucei signal sequences will be studied. Specifically, this example will test whether peptide motifs that have discovered in h-regions (i.e., h-motifs) contribute to VSG translocation into the ER. The importance of specific amino acids (e.g., Leu and Val), order of residues, and hydrophobicity of the amino acids will be examined using both in vitro and in vivo approaches.
Two trypanosome proteins (VSG—117 and VSG_MVAT7) will be used to investigate the role of h-region motifs in the import of a native T. brucei protein into the ER. Motifs will be altered by site-directed mutagenesis to determine how h-region motifs affect signal peptide function. Then, h-motifs will be studied in context of a model peptide that will enable us to test whether or not T. brucei h-motifs are important for signal sequence activity in a trypanosome.
First, this example will determine if a single motif in a natural h-region is sufficient for protein import into the ER. Second, this example will explore whether the positioning of hydrophobic amino acids in a motif is sufficient for signal peptide activity, based on the observations that the all the fixed and ambiguous components in the T. brucei motifs are hydrophobic amino acids, although T. brucei signal sequences have many polar residues (e.g., Ser and Thr). It is predicted that any three hydrophobic residues (from the group Ala, Met, Cys, Phe, Leu, Val or Ile) could be substituted for the fixed and ambiguous components of the motif without diminishing biological activity of the signal peptide. Third, this example will investigate whether the order of amino acids in a motif is important for biological function. It is predicted that scrambling the h-motif sequence will either inhibit or abolish import of protein into the ER. Fourth, this example will evaluate the importance of h-motifs for variant surface glycoprotein (VSG—117) translocation into the ER in vivo.
Full-length VSG—117 containing the h-region mutants or the unmutated signal sequence will be tested for translocation into TbRM and the data quantitated. For each cargo substrate, the relative efficiency of VSG import into TbRM will be normalized to the activity of the unmutated VSG—117 h-region, in order to assess any inhibitory (or stimulatory) effects of the mutations on entry of VSG—117 into the ER. For a change in VSG import to be considered significant, the decrease in protein translocation must be greater than 50%.
H-regions containing various T. brucei h-motifs (singly, and in combinations found in the two native signal peptides VSG—117 and VSG_MVAT7) will be used to replace that of VSG—117, using megaprimer oligonucleotide-directed PCR mutagenesis and a cDNA encoding VSG—117 as template. RNA will be prepared using the purified PCR products as template for transcription, and used for translation/translocation with T. brucei microsomes. The proportion of protein imported into TbRM will be quantitated by comparing the residual radiolabeled substrate after protease K treatment with input (total) radiolabeled protein.
This example will determine how different motifs in h-regions of T. brucei alone, in multiple copies, or in different combinations contribute to signal peptide activity in T. brucei. It will also determine how the different h-motifs in two native signal peptides (VSG—117 and VSG_MVAT7) affect the ability of the proteins to enter the ER of a trypanosome.
Example 7
H-Motifs: The Importance of Hydrophobic Residues in ER Signal Peptides
Protein entry into the ER lumen depends on two components, Sec61 translocon at the ER membrane and a signal peptide (reviewed be Rapoport, 2007, Nature; 450(7170):663-9). Protein translocation into the ER lumen can occur by two mechanisms. In mammals, secretory precursor proteins are brought to and translocated through the Sec61 translocon predominantly by a co-translational mechanism that requires a signal recognition particle (SRP) and its receptor (SR) (Walter and Blobel, 1981, J Cell Biol; 91(2 Pt 1):557-61; reviewed by Rapoport, 2007, Nature; 450(7170):663-9). In a second mechanism, termed post-translational translocation, protein import occurs after translation of the secretory precursor is completed in the cytoplasm. This mechanism involves the Sec71/72 and Sec62/63 complexes as well as the Sec61 translocon (reviewed by Matlack et al., 1998, Cell; 92(3):381-90; and Rapoport, 2007, Nature; 450(7170):663-9). Furthermore, cytosolic Hsp70 and Hsp40 chaperones are necessary for post-translational protein import into the ER (Caplan et al., 1992, Cell; 71(7):1143-55; Becker et al., 1996, Mol Cell Biol; 16(8):4378-86; McClellan and Brodsky, 2000, Genetics; 156(2):501-12; Ngosuwan, 2003, J Biol Chem; 278(9):7034-42; and reviewed by Rapoport, 2007, Nature; 450(7170):663-9). In both mechanisms, post-targeting recognition of a signal peptide at the Sec61 translocon is necessary for protein translocation into the ER (Jungnickel and Rapoport, 1995, Cell; 82(2):261-70). Recognition of signal peptides at the ER involves transmembrane segments TM2 and TM7 of Sec61 (Plath et al., 1998, Cell; 94(6):795-807; Van den Berg et al., 2004, Nature; 427(6969):36-44; and reviewed by Clemons et al., 2004, Curr Opin Struct Biol; 14(4):390-6).
An ER signal peptide has a charged n-region, an h-region composed of approximately 7-13 non-charged or hydrophobic amino acids, and a polar c-region (von Heijne, 1985, J Mol Biol; 184(1):99-105; Haeuptle et al., 1989, J Cell Biol; 108(4):1227-36; and reviewed by Izard and Kendall, 1994, Mol Microbiol; 13(5):765-73; and Hegde and Bernstein, 2006, Trends Biochem Sci; 31(10):563-71). The hydrophobic core is necessary for signal peptide function (Kaiser et al., 1987, Science; 235(4786):312-7), and the peak hydrophobicity of this region is a major factor governing the ability and pathway by which a protein enters the ER (Chou and Kendall, 1990, J Biol Chem; 265(5):2873-80; Doud et al., 1993, Biochemistry; 32(5):1251-6; Ng et al., 1996, J Cell Biol; 134(2):269-78; and reviewed by Hegde and Bernstein 2006).
ER signal peptide h-regions lack sequence conservation (reviewed by Zheng and Gierasch, 1996, Cell; 86(6):849-52) and some have been replaced by apparently “random” hydrophobic sequences selected to promote protein import into the ER (Kaiser et al., 1987, Science; 235(4786):312-7). Nevertheless, several studies suggest that signal peptides of equivalent peak hydrophobicity can be species-specific (Abrahmsen et al., 1985, EMBO J; 4(13B):3901-6; Bird et al., 1987, J Cell Biol; 105(6 Pt 2):2905-14; Al-Qahtani et al., 1998, Biochem J; 331 (Pt 2):521-9; Zheng and Nicchitta, 1999, J Biol Chem; 274(51):36623-30), suggesting that h-region sequences may not be composed of random hydrophobic amino acids. For example, E. coli β-lactamase and T. brucei VSG-117 h-regions are strikingly similar in composition and peak hydrophobicity, yet E. coli β-lactamase is imported into canine microsomes (CfRM) whereas T. brucei VSG-117 is not translocated into CfRM (Al-Qahtani et al., 1998, Biochem J; 331 (Pt 2):521-9). Furthermore, h-region sequences are not truly random on a gross level because all of the possible hydrophobic amino acids are not equally represented (von Heijne, 1985, J Mol Biol; 184(1):99-105; von Heijne and Abrahmsen, 1989, FEBS Lett; 244(2):439-46). Different species have preference for specific amino acids in their h-regions; Saccharomyces cerevisiae shows preference for serine while E. coli favors alanine (von Heijne, 1981, Eur J Biochem; 116(2):419-22; Nielsen et al., 1997, Protein Eng; 10(1):1-6). Finally, recent re-evaluation of h-regions from E. coli, S. cerevisiae, and humans has revealed that each species has a unique set of h-motifs in its signal peptides.
While it cannot be denied that high hydrophobicity contributes to translocation of signal peptides into the ER, this example demonstrates that peak hydrophobicity alone is not sufficient for signal peptide activity. Moreover, the arrangement of key amino acid residues (hydrophobic or polar) into motifs in an h-region (h-motifs) imparts physiological activity to a signal peptide.
Materials and Methods
Taq DNA polymerase, amino acid mixture minus methionine and cysteine, and nuclease treated rabbit reticulocyte lysate were obtained from Promega (Madison, Wis.). dNTPs were obtained from Sigma (St. Louis, Mo.). DEPC-treated water was from USB Corporation (Cleveland, Ohio); QIAquick PCR purification kit was from Qiagen (Valencia, Calif.); Ampliscribe T7 in vitro transcription kit was from Epicentre (Madison, Wis.); TRAN35S—LABELTM No-thaw metabolic labeling reagent was from MP Biomedicals (Solon, Ohio); cycloheximide was from Calbiochem (La Jolla, Calif.); proteinase K and leupeptin were from Roche (Indianapolis, Ind.); PMSF was from Boehringer Mannheim (Mannheim, Germany); ethanol and urea were from Fisher (Norcross, Ga.); Acrylagel and Bis-Acrylagel were from National Diagnostics (Atlanta, Ga.); TEMED and ammonium persulfate were from Bio-Rad (Hercules, Calif.); DE52 was from Whatman (Hillsboro, Ohio). All other chemicals and reagents were obtained from Sigma (St. Louis, Mo.).
Plasmids. All VSG-117 mutants were made through PCR mutagenesis (Inis et al., 1988, Proc Natl Acad Sci USA; 85(24):9436-40) of a plasmid template, pVSG-117 (Bangs et al., 1996, J Biol Chem; 271(31):18387-93).
Cell Strain and Isolation. Monomorphic Trypanosoma brucei strain 427 were grown in and purified from Sprague Dawley rats, using chromatography on DE52 (Cross, 1975, Parasitology; 71(3):393-417).
Primer Construction For VSG-117 h-Region Mutants. PCR mutagenesis was used to create VSG-117 h-region mutants from the template, pVSG-117, using variations of the forward primer TAATACGACT CACTATAGGG aggagggttt ttaccATGGA CTGCCATACA AAGGAGACAC TGGGGTCACA CAATGGAGGC GATCAACGAT GttcACACTA TCAttcttcT ActtcATCAC TCCAGCG (SEQ ID NO:3). Each primer contained a T7 promoter (upper case) and a translational enhancer (lower case) (Teilhet et al., 1998, Gene; 222(1):91-7). The coding region of VSG-117 starts at position 36 and ends at primer position 83; it includes the n-region of VSG-117. This is followed by primer positions 84-128, which encodes for the h-region of the VSG-117 protein. All VSG-117 h-region mutants contained mutations within the forward primer as exemplified above in the (Phe)4-VSG-11786 mutant. All VSG-117 proteins were truncated to 86 amino acids, using the reverse primer cgaacaacga aggggttcTT ATAGTGCGTA GATCGTAGCT TCGTTTC (SEQ ID NO:4), which has a stop codon after the 27 nucleotides priming for nucleotides 295-321 of the pVSG-117 sequence.
Generation of VSG-117 Mutant DNA. VSG-117 PCR products were generated in 100 μl reactions mixtures containing 224 ng pVSG-117 template, 0.5 μM of each forward and reverse primer, Innis buffer (10 mM Tris-HCl pH 8, 2.5 mM MgCl2, 0.05% Tween-20, 0.05% nonidet P-40, 50 mM KCl) (Innis et al., 1988, Proc Natl Acad Sci USA; 85(24):9436-40), 250 μM dNTPs, and 5 units of Taq DNA polymerase. PCR reactions were carried out for 25 cycles as follows: 95° C. for 90 seconds, 56° C. for 90 seconds, and 74° C. for 2 minutes. All PCR products were purified using a QIAquick PCR purification kit (Qiagen). Purified PCR products were quantitated at 260 nm.
In Vitro Transcription. One μg of purified DNA was used as a template for transcription with an AmpliscribeTM T7 kit (Epicentre Technologies). One μl (1 MBU) of RNase free DNase I was added to the reaction (20 μl) and allowed to incubate at 37° C. for 15 minutes. The mixture was extracted with an equal volume of TE-saturated phenol/chloroform, and the aqueous phase extracted with chloroform. To the aqueous phase, sodium acetate (27 mM final concentration) was added along with 2.5 times the total volume of 100% ethanol. This mixture was allowed to precipitate at −20° C. overnight. The precipitate was recovered at 16,100×g at 4° C. for 15 minutes, rinsed with 70% ethanol, and pelleted at 16,100×g. The pellet was allowed to air dry, and resuspended in 40 μl of nuclease free water. The RNA concentration was determined by measuring the absorbance at 260 nm.
Microsome Preparation from Bloodstream Form Trypanosoma brucei. Bloodstream form T. brucei lister 427 (1×1010 cells/ml) were resuspended in 5 ml of fresh homogenization buffer (HB) (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1 μg/ml TLCK, 5 μg/ml leupeptin, 0.5 mM PMSF). In a clean, pre-chilled dounce homogenizer 2.5 ml of the resuspended cells were lysed on ice by 2 repetitions of 40 strokes with a tight-fitting pestle, with a one minute break after 40 strokes. The homogenates were pooled, aliquoted into microcentrifuge tubes (1 ml/tube), and centrifuged at 400×g for 10 minutes at 4° C. The supernatants from all tubes were pooled, aliquoted into a new microcentrifuge tube, and centrifuged at 13,400×g for 20 minutes at 4° C. The supernatants from the 13,400×g centrifugation step were recovered and pooled. Pellets from the 13,400×g centrifugation step were resuspended in 50 μl/pellet (total volume) of fresh rough microsome buffer (RMB) (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 1 mM DTT, 0.5 μg/ml TLCK, 2.5 μg/ml leupeptin). The microsome concentration was determined by measuring the absorbance at 260 nm, and the concentration was adjusted to an OD260 of 50 (1 equivalent=OD260 of 50=1 μl). The microsomes were aliquoted into 20 μl portions, quick frozen in liquid nitrogen, and stored at −80° C. (pellets from the 400×g centrifugation and the supernatant from the 13,400×g centrifugation were also saved).
In Vitro Translation and Import of VSG Into T. brucei Microsomes (TbRM). RNA (250-500 ng) in DEPC-treated H2O (7 μl) was incubated at 65° C. for three minutes. After cooling on ice for one minute, 1.5 μl (75 μM final concentration) of an amino acid mixture lacking methionine and cysteine (Promega), 1.5 μl of 35S-cysteine and methionine (2.5 mCi total activity, MP Biomedicals), and 10 μl of rabbit reticulocyte lysate (Promega) was added to the RNA mixture, bringing the final volume to 20 μl. This reaction mixture was incubated at 37° C. for 1 hour. Translation was stopped with cycloheximide (1 mM final concentration).
The translation reaction was then divided into four separate portions of 5 μl each. Two of these portions were left untreated while the remaining two portions were supplemented with 1 μl (1 μl=1 equivalent, which has an OD260 of 50) of TbRM. The four reaction aliquots were further incubated at 37° C. for 1.5 hours and then transferred to ice. Next, one of each sample (translation mixture with or without TbRM) was treated with a proteolysis mixture containing urea (3M) and proteinase K (0.5 mg/ml) (final concentrations) on ice for 1 hour. PMSF (34 mM final concentration) was added to quench the proteolysis reaction. Proteins were precipitated with 60% (NH4)2SO4 on ice for 15 minutes, and centrifuged at 16,100×g at 4° C. for 7 minutes. The pellets were resuspended in 20 μl of 2.5×SDS sample buffer and resolved by SDS-PAGE in a Tris-Tricine gel system (16%—Resolving; 3%—Stacking) (as reviewed by Gallagher, 2007, “One-dimensional SDS gel electrophoresis of proteins.” Curr Protoc Cell Biol Chapter 6). Radioactive polypeptides were detected with a phosphorimager; bands were quantitated with Quantity-One software (version 4.6.5, Bio-Rad).
Quantitation of Percent VSG Import. Using Quantity-One software (version 4.6.5, Bio-Rad), phosphorimages were adjusted so that gel bands corresponding to proteinase K-protected VSG-11786 were visible. Next, background noise signals were subtracted using the “filter wizard” application. A Tiff image preserving these adjustments to the gel image was acquired. From this Tiff image, gel bands in each lane were quantitated by measuring the volume (counts*mm2) of the band representing VSG-11786. For background signal, the volume of an area in each lane of the gel image that best represented the average background pixel intensity was measured. In band and background intensity quantitation, the dimensions of the volume boxes were maintained. Next, a volume analysis report was performed to calculate the “adjusted volumes” (volume of VSG-11786 band−volume of average background bands) of the VSG-11786 bands. VSG import percentages were obtained as follows: the “adjusted volume” corresponding to a band detected in the presence of TbRM and challenged with proteinase K was divided by the “adjusted volume” corresponding to a band detected in the presence of TbRM without proteinase K. This quotient was multiplied by 100 to obtain a percentage of VSG import.
Results
Factors Other Than h-Region Peak Hydrophobicity May Govern Protein Import into ER Microsomes. The hydrophobic core (h-region) of a signal peptide is necessary for protein import into the ER (as reviewed by Hegde and Bernstein, 2006, Trends Biochem Sci; 31 (10):563-71). Surprisingly, h-region sequences are not conserved. It has been suggested that an h-region is composed of random hydrophobic amino acids, and that the overall hydrophobic nature of this region governs signal peptide activity. However, not all h-regions of similar hydrophobicity are equivalent in function (reviewed by Zheng and Gierasch, 1996, Cell; 86(6):849-52; and Hegde and Bernstein, 2006, Trends Biochem Sci; 31(10):563-71). Therefore, this example revisited the role of h-region hydrophobicity in protein import into the ER with the hope of offering new perspectives. With this example, the role of h-region hydrophobicity in ER protein import in T. brucei was evaluated using a truncated VSG-1177 substrate (86 amino acids long) and a T. brucei cell-free translocation system. VSG-117 is imported into TbRM, but it is not imported into canine microsomes, although VSG-117 possesses similar h-region hydrophobicity as other proteins that are imported into canine microsomes. From this, the hypothesis was formed that h-region characteristics other than hydrophobicity may be responsible for ER protein import, and we sought to determine how an h-region directs import of VSG-117 into TbRM. To examine the importance of h-region hydrophobicity in ER protein import, h-region mutants were created from VSG-117 (VSG-117h) where residues 4, 8, 9, and 11 were simultaneously mutated from Leu4, Leu8, Leu9, Ala11 to Gly4, Gly8, Gly9, Gly11 or Ser4, Ser8, Ser9, Ser11, creating the mutants (Gly)4-VSG-117h and (Ser)4-VSG-117h, respectively (Table 1). Unmutated VSG-117h (VSG-117h) contains seven hydrophobic amino acids (Table 1), and by mutating four of these hydrophobic amino acids to the non-hydrophobic amino acids glycine or serine the peak hydrophobicity was reduced by approximately 75% from 2.08 to 0.43 or 0.30, respectively (FIG. 10B). Because of the drop in peak hydrophobicity, VSG import into TbRM was expected to decrease in both (Gly)4-VSG-11786 and (Ser)4-VSG-11786-Curiously, while only 8% of (Gly)4-VSG-11786 was imported into TbRM, 44% of (Ser)4-VSG-11786 was imported into TbRM (FIGS. 10A (panels A-C) and 10B).









TABLE 1






Position of Amino Acids




in h-region Sequence


VSG
1 2 3 4 5 6 7 8 9 10 11 12 13 14







VSG-117h
S T M L T L S L L Y  A  I  T  P



 


(Gly)4-VSG-117h
S T M G T L S G G Y  G  I  T  P


 


(Ser)4-VSG-117h
S T M S T L S S S Y  S  I  T  P


 


(Ala)7-VSG-117h
S T A A T A S A A Y  A  A  T  P


 


(Ala)6-Ile12-VSG-
S T A A T A S A A Y  A  I  T  P


117h


 


(Ala)4-VSG-117h
S T M A T L S A A Y  A  I  T  P





Amino Acid Sequences of VSG-117h, (Gly)4-VSG-117h, (Ser)4-VSG-117h, (Ala)7-VSG-117h, (Ala)6-Ile12-VSG-117h, and (Ala)4-VSG-117h.


The h-Region sequences of VSG-117h (SEQ ID NO:5), (Gly)4-VSG-117h (SEQ ID NO:6), (Ser)4-VSG-117h (SEQ ID NO:7, (Ala)7-VSG-117h (SEQ ID NO:8), (Ala)6-Ile12-VSG-117h (SEQ ID NO:9), and (Ala)4-VSG-117h (SEQ ID NO:10) are aligned.


Position 1 denotes the beginning of the h-region and position 14 marks its end.


Mutations to VSG-117h are listed in bold.






To test whether or not import of (Ser)4-VSG-11786 into TbRM was an anomaly, a new h-region mutant was created by mutating all seven hydrophobic amino acids in the VSG-117 h-region to alanine (i.e. seven alanine residues at positions 3, 4, 6, 8, 9, 11, & 12), creating (Ala)7-VSG-117h (Table 1). (Ala)7-VSG-117h has a peak hydrophobicity more than twice that of (Ser)4-VSG-117h (FIG. 10B), and was predicted to be imported into TbRM with greater efficiency than the former signal peptide. Surprisingly, (Ala)7-VSG-11786 was not imported into TbRM (1%) (FIG. 10A (panel “D”) and FIG. 10B). This data is reminiscent of studies of E. coli signal peptides; a model h-region comprised of polyalanine is non-functional (Doud et al., 1993, Biochemistry; 32(5):1251-6). However, activity was restored to this polyalanine signal peptide when more hydrophobic amino acids were introduced into the peptide. Using these data as precedent, amino acids at positions (1) 12 or (ii) 3, 6, and 12 from VSG-117h were introduced back into (Ala)7-VSG-117h, making the new h-region mutants (Ala)6-Ile12-VSG-117h or (Ala)4-VSG-117h, respectively (Table 1). Changing Ala12 in (Ala)7-VSG-117h to Ile12 in (Ala)6-Ile12-VSG-117h raised the hydrophobicity from 0.89 in (Ala)7-VSG-117h to 1.21 in (Ala)6-Ile12-VSG-117h. Likewise, mutating Ala3, Ala6, and Ala12 in (Ala)7-VSG-117h to Leu3, Leu6, and Ile12 in (Ala)4-VSG-117h increased the hydrophobicity from 0.89 in (Ala)7-VSG-117h to 1.41 in (Ala)4-VSG-117h (FIG. 10B). Interestingly, 18% of (Ala)6-Ile12-VSG-11786 was imported into TbRM while 82% of (Ala)4-VSG-11786 was translocated into TbRM (FIG. 10A (panels “E” and “F”) and FIG. 10B).
The data from translocation of (Gly)4-VSG-11786, (Ser)4-VSG-11786, (Ala)7-VSG-11786, (Ala)6-Ile12-VSG-11786, and (Ala)4-VSG-11786 showed that as peak hydrophobicity increases VSG import into TbRM did not increase correspondingly (FIG. 10B). In fact, only (Ser)4-VSG-11786 and (Ala)4-VSG-11786 were imported into TbRM in significant amounts (44% and 82% respectively). The inability of (Gly)4-VSG-11786, (Ala)7-VSG-11786, and (Ala)6-Ile12-VSG-11786 to be imported into TbRM convincingly (FIG. 10A (panels “B,” “D,” and “E”) and FIG. 10B) seems to be at odds with data suggesting that increases in h-region hydrophobicity increases ER protein import efficiency. (Gly)4-VSG-11786, (Ala)7-VSG-11786, and (Ala)6-Ile12-VSG-11786 all have h-region hydrophobicity values higher than that of (Ser)4-VSG-11786 (0.43, 0.89, and 1.21 vs. 0.3, respectively) (FIG. 10B). One would expect that h-regions with hydrophobicity values greater than 0.3 would be imported into TbRM; however, this was not observed. These data show how h-region hydrophobicity fails to predict the biological activity of a signal peptide, and do not support the idea that peak hydrophobicity is the major factor governing protein import into TbRM. Consequently, two factors that had potential to be responsible for VSG import into microsomes were investigated; kinetics of protein translocation into the ER, and arrangement of amino acids within the h-region.
Kinetics of VSG-11786 and (Ala)4-VSG-11786 Import into ThRM. How hydrophobicity of VSG-11786 (2.08 hydrophobicity units, FIG. 10B) and (Ala)4-VSG-11786 (1.41 hydrophobicity units, FIG. 10B) affected import of VSG into ThRM was investigated by evaluating the rate and extent of translocation of the two signal peptides.
A time-course of VSG import into TbRM showed that (Ala)4-VSG-11786 is translocated into TbRM at a slower rate than VSG-11786. Whereas half of the total amount of VSG-11786 is imported into TbRM within approximately 0.5 h (FIG. 11A (panel “A”) and FIG. 11B), it took one hour for half of the total amount of (Ala)4-VSG-11786 to be imported into ThRM (FIG. 11A (panel “B”) and FIG. 11B). However, both VSG-11786 and (Ala)4-VSG-11786 achieve maximum accumulation at 1.5 hours (81% and 75%, respectively) (FIG. 11A (panels “A” and “B”) and FIG. 11B). This data supports a hydrophobicity difference of 0.67 (approximately 30%) between the h-regions of VSG-11786 and (Ala)4-VSG-11786 affected the rate of VSG import up to the 60 minute time-point. At 90 minutes and beyond, only differences in extent of VSG import into TbRM are discernable. Therefore, any reduction in import of a VSG-117 h-region mutant at the 90-minute time-point is not a consequence of slower import kinetics.
Arrangement of Amino Acids in h-Region Affects VSG Import into TbRM. If peak hydrophobicity could not explain the failure to import (Gly)4-VSG-11786, (Ala)7-VSG-11786, and (Ala)6-Ile12-VSG-11786 into TbRM (FIG. 10A (panels “B,” “D,” and “E”) and FIG. 10B), what h-region characteristics influenced the process? One hypothesis to explain these data is that the arrangement of h-region amino acids is important for signal peptide activity. To test this hypothesis, new VSG-117 h-regions were designed by scrambling the sequences of (Ala)4-VSG-117h and (Ser)4-VSG-117h, producing “scrambled (Ala)4-VSG-117h” and “scrambled (Ser)4-VSG-117h”, respectively (Table 2). Scrambling the (Ala)4-VSG-117h sequence reduced peak hydrophobicity by approximately 0.3 units (24% reduction), while “scrambled (Ser)4-VSG-117h” differed from (Ser)4-VSG-117h by only 0.01 units (3% reduction) (FIG. 12A (panel “F”). Also, a variant of (Ala)4-VSG-117h was created by exchanging tyrosine at position 10 for alanine at position 9, yielding (Ala)4-Tyr9-VSG-117h (Table 2). Because (Ala)4-Tyrg-VSG-117h was created from (Ala)4-VSG-117h by switching the positions of two amino acids, the peak hydrophobicity was not affected (FIG. 12B). Since the amino acid composition of each sequence was not altered, any change in translocation competence would be attributable to amino acid sequence alterations.
Microsomal import assays comparing “scrambled” h-region mutants to their unscrambled counterparts showed a drastic reduction in the import of the “scrambled” mutants. (Ala)4-VSG-11786 was imported into TbRM at approximately 86% while only 14% of “scrambled (Ala)4-VSG-11786” was imported into TbRM (FIG. 12A (panels “B” and “C”) and FIG. 12B). Approximately 70% of (Ser)4-VSG-11786 was imported into TbRM while 27% of “scrambled (Ser)4-VSG-11786” was imported into the microsomes (FIG. 12A (panels “D” and “E”) and FIG. 12B). Similarly, 64% of (Ala)4-Tyrg-VSG-11786 was imported into TbRM, whereas 84% of (Ala)4-VSG-11786 was translocated into TbRM (FIG. 12A (panels “B” and “F”) and FIG. 12B). These data support the principle that arrangement of h-region amino acids is a crucial factor for translocation of VSG into TbRM.
Discovery of Peptide Motifs in T. brucei h-Regions. The data of this example indicated that h-region amino acid sequence was crucial to VSG import into TbRM (FIGS. 12A and 12B). However, an alignment of trypanosome signal peptides fails to unearth a consensus sequence. To resolve this conundrum, it was hypothesized that h-region amino acids formed peptide motifs that could not be discovered with sequence alignments. In an initial test of this hypothesis, 50 h-regions of secretory proteins from T. brucei were analysed. Through computational peptide pattern searches the motifs shown Table 3 were identified.
An h-region motif (h-motif) has three “identity components” and a “wild card region”. There are two types of “identity components”; a “fixed identity component” is a unique amino acid, and a “variable identity component”, which is a position that can be filled by one of a defined set of amino acids. The “wild card region” separates “identity components”; it can be one or multiple amino acids. For example, the h-motif L-x(1,2)-L-[AILV] possesses three “identity components”. The first two are “fixed identity components”, namely leucine (L), where the first leucine is separated from the second leucine by one or two “wild card” residues. The last “identity component” is a “variable identity component” that can be alanine (A) or isoleucine (I) or leucine (L) or valine (V) (i.e. [AILV]).









TABLE 2






Position of Amino Acids




in h-region Sequence


VSG
1 2 3 4 5 6 7 8 9 10 11 12 13 14







VSG-117h
S T M L T L S L L Y  A  I  T  P



 


Scrambled
A S L T A M A T S A  Y  I  T  P


(Ala)4-VSG-117h


 


Scrambled
S S L T S M S T S S  Y  I  T  P


(Ser)4-VSG-117h


 


(Ala)4-VSG-117h
S T M A T L S A A Y  A  I  T  P


 


(Ala)4-Tyr9-VSG-
S T M A T L S A Y A  A  I  T  P


117h





Amino Acid Sequences of VSG-117h, scrambled (Ala)4-VSG-117h, scrambled (Ser)4-VSG-117h, (Ala)4-VSG-117h, and (Ala)4-Tyr9-VSG-117h.


The VSG-117h (SEQ ID NO:5), scrambled (Ala)4-VSG-117h (SEQ ID NO:11), scrambled (Ser)4-VSG-117h (SEQ ID NO:12), (Ala)4-VSG-117h (SEQ ID NO:10), and (Ala)4-Tyr9-VSG-117h (SEQ ID NO:13) mutants are aligned by h-region sequences.


Position 1 denotes the beginning of the h-region and position 14 marks its end.


h-Region sequence differences in (Ala)4-VSG-117h and (Ala)4-Tyr9-VSG-117h are listed in bold.






Three h-motifs were discovered in T. brucei (L-L-x-[AILV], L-x(1,2)-L-[AILV], and L-x(2,3)-L-[AILPV]) (Table 3). Of the 50 signal peptides, 37 (74%) contained at least one T. brucei h-motif. Most of the signal peptides contain multiple T. brucei h-motifs. This is exemplified by VSG-117, whose h-region contains one of each h-motif (Table 3). These h-motifs are different in organization and “identity component” composition from those h-motifs identified in yeast, E. coli, and humans.
Experimental Data Can Be Used to Expand “Identity Components” of Bioinformatically Identified h-Motifs. Existence of h-motifs does not prove their biological relevance. Thus, h-motifs are important for signal peptide activity. To evaluate this hypothesis, the four positions necessary to form every motif in the h-region of VSG-117 were mutated. Residues at h-region positions 4, 8, 9, and 11 (Leu4, Leu8, Leu9, and Ala11, respectively) were all changed to phenylalanine or valine (residues that were not discovered as “fixed identity components” in our data set), resulting in the new h-region mutants (Phe)4-VSG-117h and (Val)4-VSG-117h, respectively (Table 4). Although the mutants lack h-motifs (Table 3), their hydrophobicities were similar to that of VSG-117h (FIG. 13B). Therefore, any reduction in protein import would be due principally to the loss of the h-motif. Both (Phe)4-VSG-11786 and (Val)4-VSG-11786 were imported into TbRM with similar efficiencies (78% and 83%, respectively) as compared to VSG-11786 (99%) (FIG. 13A (panels “A,” “B,” and “C”) and FIG. 13B. These data were compared with earlier ones (FIG. 10A (panels “B”-“F”), FIG. 10B, FIG. 13A (panels “A”-“C”) and FIG. 13B) to obtain a general picture of the contributions of h-motifs to signal peptide activity.
The signal peptides of (Ala)4-VSG-11786 and (Ser)4-VSG-11786 were efficient at directing protein import into TbRM (FIG. 12A (panels B and D) and FIG. 12B). However, scrambling the h-region sequences of (Ala)4-VSG-11786 and (Ser)4-VSG-11786 caused a drastic reduction in the ability of these signal peptides to direct import into TbRM (FIG. 12A (panels C and E) and FIG. 12B). This indicated that the arrangement of h-region amino acids is crucial for signal peptide activity. Bioinformatic analysis of T. brucei signal peptides suggests that h-region amino acids are arranged into h-motifs where specific hydrophobic amino acids serve as “identity components” (Table 3). Data from “scrambled (Ala)4-VSG-11786” and “scrambled (Ser)4-VSG-11786” combined with bioinformatic data suggests that the rearrangement of h-region amino acids in “scrambled (Ala)4-VSG-11786” and “scrambled (Ser)4-VSG-11786” disrupted the arrangement of h-region amino acids serving as “identity components” in these h-motifs. This, in turn, affected the ability of these signal peptides to direct import into TbRM. Like (Ser)4-VSG-11786 and (Ala)4-VSG-11786, (Phe)4-VSG-11786 and (Val)4-VSG-11786, were imported into TbRM efficiently (FIG. 10A (panels C and F), FIG. 10B, FIG. 13A (panels A, B, and C) and FIG. 13B) while (Gly)4-VSG-11786, (Ala)7-VSG-11786, and (Ala)6-Ile12-VSG-11786 were barely imported into TbRM (FIG. 10A (panels B, D, and E) and FIG. 10B).










TABLE 3







Location of Amino Acids





in h-Motifs of VSG-117h


Name
h-Motif
1 2 3 4 5 6 7 8 9 10 11 12 13 14









S T M L T L S L L Y  A  I  T  P



Tb1
L-L-x-[AILV]
              L L x  A


 


Tb2
L-x(1, 2)-L-
          L x L L



[AILV]


 


Tb3
L-x(2, 3)-L-
      L x x x L L



[AILPV]





h-Region Peptide Motifs in VSG-117.


The peptide motifs in the VSG-117 h-region are shown. Motifs are displayed in PROSITE syntax.


Peptide patterns were obtained from analysis of 50 T. brucei h-regions, using PRATT pattern matching tool (Jonassen et al., 1995, Protein Sci; 4(8):1587-95).


The PRATT parameters were determined empirically to identify patterns with three or more “identity components.” The parameters were: C% (52); PL (50); PN (50); PX (5); FN (5); FL (2); FP (20); E (3).


The amino acid sequence STMLTLSLLYAITP is VSG-117h (SEQ ID NO:5), Tb1 (LLxA) is SEQ ID NO:14, Tb2 (LxLL) is SEQ ID NO:15, and Tb3 (LxxxLL) is SEQ ID NO:16.






However, all VSG-117h mutants with functional signal peptides or non-functional signal peptides lack T. brucei h-motifs identified by bioinformatics (compare sequences in Table 1 and 4 with motifs in Table 3). To reconcile this dilemma the bioinformatic and experimental data were combined, and the conclusion that “identity components” are important for efficient protein import into TbRM was drawn, and only a select set of amino acids can serve as “identity components” for a signal peptide to function. However, the data of this example indicate that T. brucei h-motifs allow hydrophobic residues (or serine residues) other than those found by bioinformatics to serve as “identity components.”









TABLE 4






Position of Amino Acids




in h-region Sequence


VSG
1 2 3 4 5 6 7 8 9 10 11 12 13 14







VSG-117h
S T M L T L S L L Y  A  I  T  P



 


(Phe)4-VSG-117h
S T M F T L S F F Y  F  I  T  P


 


(Val)4-VSG-117h
S T M V T L S V V Y  V  I  T  P





Sequences of VSG-117h, (Phe)4-VSG-117h, and (Val)4-VSG-117h h-Regions.


Amino acids in VSG-117h (SEQ ID NO:5), (Phe)4-VSG-117h (SEQ ID NO:17), and (Val)4-VSG-117h (SEQ ID NO:18) are aligned by h-region sequences.


Position 1 is the beginning of the h-region and position 14 marks its end.


Mutations in the VSG h-regions are listed in bold.






VSG-117 h-Regions do Not Require Leucines. Mammalian h-regions are leucine-rich (von Heijne, 1981, Eur J Biochem; 116(2):419-22; von Heijne and Abrahmsen, 1989, FEBS Lett; 244(2):439-46; and Izard and Kendall, 1994, Mol Microbiol; 13(5):765-73). Experimental demonstration of a requirement for a leucine in a mammalian h-region was obtained in a study of E. coli proLamB. proLamB was non-functional at the canine microsome, but gained translocation activity into CfRM when leucine was substituted for alanine residues in its h-region (Zheng and Nicchitta, 1999, J Biol Chem; 274(51):36623-30). In an earlier study, replacement of glycine with leucine in the h-region of S. cerevisiae carboxypeptidase Y allowed the yeast protein to be imported into canine microsomes (Bird et al., 1987, J Cell Biol; 105(6 Pt 2):2905-14). The importance of leucine in trypanosome signal peptides has not been tested directly.









TABLE 5






Position of Amino Acids




in h-region Sequence


VSG
1 2 3 4 5 6 7 8 9 10 11 12 13 14







(Ala)4-VSG-117h
S T M A T L S A A Y  A  I  T  P



 


(Ala)4-Phe6-VSG-
S T M A T F S A A Y  A  I  T  P


117h


 


(Ala)4-Val6-VSG-
S T M A T V S A A Y  A  I  T  P


117h


 


(Ala)4-Ile6-VSG-
S T M A T I S A A Y  A  I  T  P


117h





Amino Acid Sequences of (Ala)4-VSG-117h, (Ala)4-Phe6-VSG-117h, (Ala)4-Val6-VSG-117h, and (Ala)4-Ile6-VSG-117h.


The h-Region sequences of (Ala)4-VSG-117h (SEQ ID NO:10), (Ala)4-Phe6-VSG-117h (SEQ ID NO:19), (Ala)4-Val6-VSG-117h (SEQ ID NO:20, and (Ala)4-Ile6-VSG-117h (SEQ ID NO:21) are aligned.


Position 1 marks the beginning of the h-region and position 14 is its end.


Mutations to the VSG h-regions are listed in bold.






The majority of VSG-117 signal peptide variants that were imported into TbRM possessed at least one leucine in their h-regions. However, data from (Gly)4-VSG-11786, “scrambled (Ala)4-VSG-11786”, and “scrambled (Ser)4-VSG-11786” showed that leucine is not sufficient for efficient VSG import into TbRM because each of these h-regions contained leucine, yet they were imported into TbRM in negligible amounts (FIG. 10A (panel “B”), FIG. 10B, FIG. 12A (panels “C” and “E”) and FIG. 12B). Nevertheless, leucine is found in all h-motifs identified in T. brucei (Table 3). From these facts we hypothesized that while leucine is not sufficient for signal peptide activity, it could facilitate efficiency of h-region activity at TbRM. To investigate this idea new variants of (Ala)4-VSG-117h were made where its sole leucine at position six was mutated to other hydrophobic amino acids, namely, phenylalanine, valine, or isoleucine, creating the mutants (Ala)4-Phe6-VSG-117h, (Ala)4-Val6-VSG-117h, and (Ala)4-Ile6-VSG-117h, respectively (Table 5). Since (Ala)4-VSG-117h, (Ala)4-Phe6-VSG-117h, (Ala)4-Val6-VSG-117h, and (Ala)4-Ile6-VSG-117h only differed by one amino acid at position six their h-region peak hydrophobicity values were similar (FIG. 14B). Therefore, any difference in the extent of VSG import among these h-regions is not attributable to peak hydrophobicity.
The h-regions were imported into TbRM with different efficiencies; import efficiencies for (Ala)4-Phe6-VSG-11786, (Ala)4-Val6-VSG-11786, and (Ala)4-Ile6-VSG-11786 were 71%, 54%, and 40%, respectively (FIG. 14A (panels “A,” “B,” “C,” “D,” and “E”) and FIG. 14B). Therefore, while mutating leucine to phenylalanine altered VSG import efficiency into TbRM by 6%, substitution of leucine with valine or isoleucine caused significant reduction (14% or 28%, respectively) in the amount of VSG imported into TbRM (FIG. 14B).
This example demonstrates that leucine in the VSG-117 h-region is not essential for signal peptide activity; other hydrophobic residues can be used in the signal sequence. Further, as the hydrophobicity of the amino acid occupying position six of (Ala)4-VSG-117h increased the efficiency of import decreased (FIG. 14A (panels “A,” “B,” “C,” “D,” and “E”) and FIG. 14B, suggesting a hierarchy of hydrophobic residues (L≧F>V>I) in that position of the h-region.
Discussion
Arrangement of h-Region Amino Acids Is Important For Signal Peptide Function. Cell surface protein expression is essential for cell viability in all biological kingdoms. Instrumental to entry into the secretory pathway is protein translocation into the ER (reviewed by Shimizu and Hendershot, 2007, Adv Exp Med Biol; 594:37-46), a process that requires an ER signal peptide (reviewed by Schatz and Dobberstein, 1996, Science; 271(5255):1519-26; and Hegde and Bernstein, 2006, Trends Biochem Sci; 31(10):563-71). To date the best model available to explain signal peptide function relies heavily on h-region peak hydrophobicity as the determinant of activity (Ryan et al., 1986, J Biol Chem; 261(7):3389-95; Chou and Kendall, 1990, J Biol Chem; 265(5):2873-80; Doud et al., 1993, Biochemistry; 32(5):1251-6; Ryan et al., 1993, Virus Genes; 7(1):5-21; Tomilo et al., 1994, J Biol Chem; 269(50):32016-21; and Ng et al., 1996, J Cell Biol; 134(2):269-78). Although many experiments support this paradigm, there are numerous exceptions to this dogma (Abrahmsen et al., 1985, EMBO J; 4(13B):3901-6; Bird et al., 1987, J Cell Biol; 105(6 Pt 2):2905-14; Laforet et al., 1989, J Biol Chem; 264(24):14478-85; von Heijne and Abrahmsen, 1989, FEBS Lett; 244(2):439-46; Ryan and Edwards, 1995, J Biol Chem; 270(46):27876-9; Al-Qahtani et al., 1998, Biochem J; 331 (Pt 2):521-9; Matoba and Ogrydziak, 1998, J Biol Chem; 273(30):18841-7; and Ramirez et al., 1999, J Eukaryot Microbiol; 46(6):557-65). These observations bolster two ideas; one, h-region hydrophobicity alone is not sufficient to impart signal peptide function, and, two, the h-region sequence contains cryptic information that is crucial for signal peptide activity.
Three sets of data show that h-region peak hydrophobicity cannot predict import of VSG-117 h-region variants into TbRM (FIGS. 10A and 10B). First, introduction of four serine residues into specified positions of the VSG-117 h-region (Table 1) produced an h-region with one of the lowest peak hydrophobicity values in this study (FIG. 10B), yet it was imported into TbRM (FIG. 10A (panel “C”) and FIG. 10B). In contrast, (Gly)4-VSG-11786 was imported into TbRM in negligible amounts even though the peak hydrophobicity of this h-region was very similar to that of (Ser)4-VSG-11786 (FIG. 10B). Further, (Ala)7-VSG-11786 and (Ala)6-Ile12-VSG-11786 possessed even higher h-region peak hydrophobicities, but were not translocated convincingly (1% and 18%, respectively) (FIG. 10A (panels “D” and “E”) and FIG. 10B). Second, import of “scrambled (Ser)4-VSG-11786” (FIG. 12A (panels “D” and “E”) and FIG. 12B) was the most striking example of how h-region peak hydrophobicity does not solely govern signal peptide function. Peak hydrophobicity of “scrambled (Ser)4-VSG-117h” differed by only 0.01 from (Ser)4-VSG-117h, yet 27% of “scrambled (Ser)4-VSG-11786” was imported into TbRM while 70% of (Ser)4-VSG-11786 was imported into TbRM (FIG. 12A (panels “D” and “E”) and FIG. 12B). Third, exchanging amino acids at positions Ala9 and Tyr10 in (Ala)4-VSG-11786 for Tyr9 and Ala10 in (Ala)4-Tyrg-VSG-11786 (Table 2) did not alter the peak hydrophobicity of (Ala)4-VSG-11786 and (Ala)4-Tyrg-VSG-11786, but caused a significant reduction in import (22%) of (Ala)4-Tyrg-VSG-11786 into ThRM (FIG. 12A (panels “B” and “F”) and FIG. 12B). These data make a compelling case that peak hydrophobicity of the h-region is not the sole determinant of signal peptide activity.
h-Regions Contain Amino Acid Motifs That Are Important for Biological Function of Signal Peptides. To discover what other information, apart from hydrophobicity, that might be present in h-regions this example used bioinformatic approaches (Jonassen et al., 1995, Protein Sci; 4(8):1587-95) to analyze h-regions from T. brucei, and discovered three motifs (Table 3). This observation supports the idea that h-region sequences are not random, as previously believed (reviewed by Zheng and Gierasch, 1996, Cell; 86(6):849-52).
The functional significance of h-motifs was evaluated experimentally by mutating “identity component” positions 4, 6, 8, 9, and 11 (Tables 1, 4, and 5). The data showed that hydrophobic amino acids at positions 4, 8, 9, and 11 could be replaced with other hydrophobic amino acids and also with serine (FIG. 10A (panels “B,” “C,” and “F”), FIG. 10B, FIG. 13A (panels “B,” and “C”) and FIG. 13B). Further, leucine at h-region position six could be functionally replaced with phenylalanine, valine, or isoleucine (FIG. 14A (panels “A,” “B,” “C,” “D,” and “E”) and FIG. 14B). However, hydrophobicity of the amino acid occupying this position appeared to be inversely proportional to efficiency of VSG import for that h-region. This begs the question that if other hydrophobic amino acids and serine can serve as identity components why were they not found in the bioinformatic analysis? One possible explanation as to why h-motifs containing phenylalanine, valine, isoleucine, or serine were not identified (Table 3) is that proteins containing them were not a significant proportion of the T. brucei proteome. The bioinformatic analysis of this example would not score motifs from small sets because they did not comprise 52% (PRATT minimum percentage (C %)) of the total input data set.
Natural selection and signal peptide competition for ER entry could also explain why some h-motifs direct VSG import in vitro, but are not detected in trypanosome signal peptides. Mutations could have created h-region sequences that were preferred by the translocation machinery over other h-regions. Thus, while they still directed import in vivo these less efficient h-region sequences might have been out-competed by the more efficient signal peptides. Similarly, an example of signal peptide competition in E. coli showed that advantageous mutations in a signal peptide corresponded with preferential use of this more efficient signal peptide over its non-mutated counterpart to direct protein translocation (Chen et al., 1996, J Bacteriol; 178(23):6658-64). Alternatively, h-region sequences that direct import in vitro might be too efficient in vivo, out-competing signal peptides that direct import of proteins essential for viability, possibly causing cells to be non-viable. As such, these signal peptides would not be preserved during evolution.
Collectively, this example supports the arrangement of hydrophobic amino acid within an h-region is crucial for the biological activity of a signal peptide. This example demonstrates that h-region amino acids are arranged into motifs, and it is the presence of these h-motifs that direct protein translocation into the ER. In this scenario, h-regions are hydrophobic because they contain h-motifs that possess predominantly hydrophobic “identity components.” The elucidation of h-motifs in signal peptides indicates that primary sequence has a role in signal peptide activity.
h-Motifs Interact With TM2b and/or TM7 of Sec61α. Signal peptides are proposed to have a high propensity to form α-helices (Bruch and Gierasch, 1990, J Biol Chem; 265(7):3851-8; Izard and Kendall, 1994, Mol Microbiol; 13(5):765-73). From chemical cross-linking studies in S. cerevisiae, and X-ray crystal structure of Sec61 it has been proposed that transmembrane domains (TMs) 2b and 7 of Sec610 are in proximity to signal peptides (Plath et al., 1998, Cell; 94(6):795-807; Van den Berg et al., 2004, Nature; 427(6969):36-44). However, it is not known what areas of signal peptide h-regions are contacted by Sec61α. With this example, helical-wheel modeling of the VSG-117 h-region was used to offer a structure-based explanation of how h-motifs might interact with the Sec61 translocon and confer biological function to a signal peptide.
T. brucei h-motifs Tb1-3 (Table 3) specify positions 4, 6, 8, 9, and 11 of the VSG-117 h-region as “identity components.” When the VSG-117 h-region is arranged into an α-helix, the “identity components” at h-region positions 4, 8, and 11 are placed on one side of an α-helix. Interestingly, the “identity components” at h-region positions 6 and 9 are positioned on the other half of the α-helix of the h-region (FIG. 15). Such positioning of h-motif “identity components” could allow amino acids at those positions to make concurrent contacts with transmembrane helices TM2b and/or TM7 of Sec61α (FIG. 51 and FIGS. 16A-16D) (Van den Berg et al., 2004, Nature; 427(6969):36-44). Chemical cross-linking studies and a Sec61-signal peptide model from yeast (Plath et al., 1998, Cell; 94(6):795-807) suggests that h-region amino acids on opposite sides of the signal peptide α-helix could make three contacts with TM2b of Sec61α and two contacts with TM7 of Sec61α. Using this as a model, it is apparent that the three h-region “identity components” in positions 4, 8, and 11 contact one half of the signal peptide-binding site of Sec61 (possibly at TM2b) while the two h-region “identity components” in positions 6 and 9 contact the other half of the Sec61α signal peptide-binding site (possibly at TM7) (FIGS. 16A-16D).
An ER signal peptide was once thought to be a random set of hydrophobic residues, but as supported by this example, turns out to be unexpectedly more complex than originally anticipated. Hydrophobic cores are non-charged “scaffolds” in which h-motifs, important for signal peptide function, are embedded.
Example 8
Drug Discovery with TbRM
The TbRM system of the present invention will advance our understanding of structure and function of trypanosome signal peptides that are important for targeting of nutrient receptors, virulence factors, and variant surface antigens to the plasma membrane of the parasite.
Using the methods described in more detail in the previous examples, which identified equisetin, CJ21,058, and MAL3-101 as new lead compounds for anti-trypanosome drug discovery, additional compounds will be screened, to identify additional lead compounds.
Example 9
Preparation of Microsomes from Other Parasitic Organisms
Using the methods described in more detail in Examples 1-6, for the preparation of microsomes from trypanosomes, microsomes will be prepared from other protozoan parasites, including, various Leishmania species, such as, for example, Leishmania major. Such microsomes may be used for the characterization of signaling sequences in the transport and translocation of proteins in protozoan parasites and in assays for the identification of lead compounds for the development of new drugs for the treatment and prevention of diseases caused by various parasitic organisms.
Example 10
A Full Length VSG—117 Construct and Drug Discovery at the Endoplasmic Reticulum of a Trypanosome
With this example, a trypanosome microsomal system (TbRM) that imports full length VSG—117 post-translationally was developed. Using this system, MAL3-101, equisetin and CJ-21,058 functioned as small molecule inhibitors of VSG—117 translocation into the ER. These agents also killed blood stream T. brucei in vitro; the concentrations at which fifty percent of parasites was killed (IC50) were 1.5 μM (MAL3-101), 3.3 μM (equisetin) and 7 μM (CJ-21,058), respectively. Thus, VSG—117 import into ThRM is a rapid and novel assay to identify “new chemical entities” for anti-trypanosome drug development and MAL3-101, equisetin, and CJ-21,058 as new lead compounds for anti-trypanosome drug discovery.
Materials and Methods
Reagents and Chemicals. Plasmid pVSG—117 (Bangs et al., 1996, J Biol Chem; 271(31):18387-93) served as a temple. The acyl tetramic acid (3-acyl-5-hydroxymethyl-2,4-dione) derivatives CJ-21,058 and equisetin were from Pfizer Inc. (New York, N.Y.). Rabbit reticulocyte lysate and methionine/cysteine-free amino acid mixture was purchased from Promega (Madison, Wis.). [35S]Redivue PromixTM was purchased from Amersham Biosciences (Piscataway, N.J.), etoposide from Sigma (St. Louis, Mo.). Ampliscribe T7 In vitro transcription kit was purchased from Epicentre Technologies (Madison, Wis.).
DNA Templates and RNA Synthesis In vitro. DNA template (one μg) was transcribed using the AmpliscribeTM T7 kit (Epicentre Technologies) following the manufacturer's protocol. A template for in vitro transcription was obtained by PCR using the forward primer ccctaatacg actcactata gggaggaggg tttttaccat ggactgccat acaaaggag (SEQ ID NO:1), which contains a T7 promoter and a translation enhancer. The first 21 nucleotides of the VSG—117 coding sequence are in regular style font. The forward primer for VSG—117500ΔSP was ccctaatacg actcactata gggaggaggg tttttaccat ggccacactg agaaaggttgc (SEQ ID NO:24). It contained nucleotides 51-57 of the coding region. Other features are similar to those mentioned for the previous forward primer. The reverse primer for VSG—117500 was catcatcatt tcctaaaaaa gcaaggc (SEQ ID NO:25). It contained nucleotides 516-525 of the coding region. The substrate for synthesis of VSG_MVAT7 was plasmid SL/3 (kindly provided by Dr. John Donelson, Univ. of Iowa).
The forward primer for the reaction was ccctaatacga ctcactatagg gaggagggtt tttaccatgt caacaagagt ccaacaa (SEQ ID NO:26). It contained a T7 promoter, a translational enhancer, and the first 21 nucleotides of VSG_MVAT7 coding sequence. One μl (1 MBU) of RNase free DNase I was added to the reaction mixture that was incubated at 37° C. for 15 minutes (1 MBU digests 1 μg of pUC19 DNA in 10 minutes (“min”)). An equal volume of TE-saturated phenol/chloroform was then added to the reaction, which was agitated on a Vortex mixer, and centrifuged (13,200 rpm, 15 minutes) at 4° C. The aqueous phase was withdrawn, combined with an equal volume of chloroform, agitated on a Vortex mixer, and centrifuged (15 minutes, 13,200 rpm, 4° C.). To this aqueous phase 0.3M sodium acetate (final concentration) and precipitated with ethanol at −20° C. overnight. The precipitate was recovered by centrifugation (13,200 rpm, 4° C. for 15 minutes), rinsed with 70% ethanol, air-dried and dissolved 40 μl of nuclease free water. The RNA concentration was determined by measuring the absorbance at 260 μm, and confirmed by analysis with agarose gel electrophoresis/ethidium bromide staining.
Preparation of T. brucei Microsomes (TbRM). Blood stream trypanosomes were obtained from the blood of infected rats, and purified by DE-52 chromatography. Cells (1×1010) were resuspended in 5 ml of homogenization buffer (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 1 μg/ml TLCK, 5 μg/ml leupeptin, 0.5 mM PMSF (final concentrations). Two and half ml of resuspended cells were added to a pre-chilled dounce homogenizer where they were lysed with 40 strokes of a tight-fitting pestle on ice. After a 1 minute rest, another after 40 strokes was delivered with the pestle. Homogenized cells were kept on ice while the remaining 2.5 ml of resuspended cells were broken. Both sets of homogenized lysates were pooled and centrifuged (2,000 rpm, 10 min, 4° C.) in microfuge tubes. The supernatants were pooled, aliquoted into new microfuge tubes, and centrifuged at 12,000 rpm for 20 min at 4° C., and pellets were resuspended in 40 μl (total volume) of fresh rough microsome buffer (RMB) (250 mM sucrose, 50 mM HEPES-KOH, 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM DTT, 0.5 μg/ml TLCK, 2.5 μg/ml leupeptin). The absorbance of the resuspended pellet (TbRM) was determined at 260 nm, and the concentration of TbRM was adjusted (with RMB) to OD260 nm of 50. TbRM was aliquoted (20 μl portions), quick frozen in liquid nitrogen, and stored at −80° C. One equivalent of TbRM has an OD260 of 50. Supernatant from the 12,000 rpm centrifugation step (above) was also saved (see below).
Preparation of Cytosol from T. brucei. The 12,000×g supernatant obtained during the preparation of TbRM (see above) was centrifuged at 65,000×g (60 min, 4° C., Beckman TLA 100.3 rotor). Two ml of the resulting supernatant was concentrated twenty-fold by ultrafiltration with a Centricon-10 filter (Amicon). The retentate was retrieved, an aliquot was diluted 50-fold with 0.1% SDS, and the OD280 was measured. One equivalent of cytosol has an OD280 of 50. Fifty microliter aliquots were quick-frozen in liquid nitrogen, and stored at −80° C.
Protein Import Into TbRM. Two μg of RNA encoding VSG—117 (or VSG_MVAT7) was translated in 30 μl of a reaction mixture containing 15 μl rabbit reticulocyte lysate, 60 μM amino acid mixture (-Met, -Cys), and 2.4 μCi [35S]Promix. The reaction was incubated at 37° C. for 15 min. Cycloheximide (50 μg/ml, final concentration) was used to stop further translation, and the mixture was divided into two portions. T. brucei microsomes (TbRM) (1 equivalent) was added to one aliquot, and to the other portion an equal volume of RM buffer was added. The reactions were incubated at 37° C. for 45 min. To measure the post-translational import of VSG—117 into TbRM, each reaction mixture was divided into three portions (each 10 μl) and treated with one of the following on ice for one hour: (i) RM buffer; (ii) 30 μg/ml protease K; (iii) NP-40 (2%, final concentration) and protease K (30 μg/ml, final concentration). PMSF (20 mM final concentration) was added to stop protease K digestion. Samples were precipitated with an equal volume of cold (NH4)2SO4 sulphate (saturated) and total proteins were resolved by SDS-PAGE (14% total acrylamide, 3% cross linker in Tricene-HCl system). The gels were dried, and radioactive polypeptides were detected with a phosphorimager (Personal Molecular Imager FX, (BioRad). Data were quantified with QuantityOne software (BioRad), and graphs were plotted with DeltaGraph (Red Rock software).
Results
T. brucei Microsomes (TbRM) Import Trypanosome VSGs. Microsomes from canine pancreas are a model system used to study protein import into the ER of eukaryotes. Unfortunately, signal sequences of T. brucei are generally incompatible with the protein import machinery of canine microsomes. Consequently, it was crucial to develop a trypanosomatid microsome system using ER membranes from T. brucei (TbRM) in order to study the ER import of a trypanosome protein. The substrate used for these studies was the VSG coat protein. Standard tests for successful microsomal ER protein import include: proteinase protection of the translocated protein, and loss of protease protection after detergent permeabilization of the microsomes. Native VSG protein is retained on membranes by a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. All VSG constructs were engineered to produce “soluble secreted proteins” by addition of a stop codon upstream of the coding sequence for the GPI-signal peptide. Following import into ER microsomes, a soluble secreted protein is protected from exogenous protease by the ER membrane.
In this example, full-length VSG—117 was tested as a substrate protein for TbRM (see FIG. 17A for a flow chart of the protocol). In Example 2, truncated VSG—11786 (86 amino acids long) was translocated in ThRM. However, full-length VSG—117 (500 amino acids long) was not imported into the microsomes (FIG. 17, lanes 1-3). Cytosolic chaperones that enable full-length proteins to maintain an “import-competent status” are required for post-translational import of proteins into the ER of yeasts. Therefore, it was suspected that full-length VSG—117 was not imported into TbRM because trypanosome chaperones were absent from the reaction mixture of reticulocyte lysate and TbRM. Thus, cytosol was prepared from T. brucei and evaluated its effect on translocation of full-length VSG—117 into TbRM.
Addition of T. brucei cytosol to TbRM led to protection of VSG—117 from protease K digestion (FIG. 17B, lanes 4 and 5). Detergent permeabilization of TbRM allowed proteinase K to digest VSG—117 in presence of cytosol (FIG. 17A, lane 6), indicating that the proteaseprotected full-length VSG—117 was imported into TbRM. Thus, cytosol from T. brucei is required for import of full-length VSG—117 into TbRM.
To test whether results with VSG—117 were applicable to other trypanosome proteins, this example also tested whether TbRM could import another full-length trypanosome protein VSG_MVAT7 (500 amino acids). This substrate was chosen because the VSG_MVAT7 signal peptide primary structure is different from that of VSG—117 (Al-Qahtani et al., 1998, Biochem J; 331 (Pt 2):521-9). Nevertheless, in the absence of TbRM, VSG_MVAT7 was degraded by proteinase K (FIG. 17C, lane 2), but the addition of cytosol and TbRM led to protection of VSG_MVAT7 from protease K (FIG. 17C, lane 5). Without cytosol the protein was not imported in TbRM. When detergent was introduced, proteinase K degraded the protected VSG_MVAT7 (FIG. 17, lane 6). Thus, VSG_MVAT7 is also imported into TbRM.
N-terminal signal sequences mediate import of proteins into the ER. Therefore the role of a signal sequence in VSG translocation into TbRM was evaluated with VSG—117500ΔSP, which was obtained by deleting the signal peptide from VSG—117. VSG—117500ΔSP was not protected from proteinase K in the presence of both ThRM and cytosol from T. brucei (FIG. 17D, compare lanes 3 and 4). Thus, an N-terminal signal peptide is essential for VSG—117 import into TbRM.
Proteins imported into the TbRM were not N-glycosylated because the molecular weight of the protease-protected VSGs was identical to the size of the protein synthesized in the absence of TbRM (FIG. 17B and FIG. 17C). However, absence of glycosylation does not mean that TbRM failed to import the proteins. Protein import into the ER and subsequent processing can be uncoupled, with oligosaccharyltransferase (OST) and signal peptidase acting after import of the substrate by the Sec61p complex. Thus it is likely that TbRM is deficient in OST activity, as has been observed in other in vitro ER systems. Nevertheless, the ThRM import assay of the present invention meets the “gold standard” for demonstrating the movement of a polypeptide into a membrane vesicle (or organelle), protease protection of the imported cargo, and digestion of the previously protected cargo by protease in the presence of detergent that permeabilizes the microsomal membrane.
MAL3-101 Inhibits VSG Translocation into TbRM. Post-translational protein import into S. cerevisiae ER and across the E. coli inner membrane requires cytosolic chaperones or chaperone-like proteins, respectively. Since import of full-length VSG into T. brucei microsomes is posttranslational and dependent on cytosol, the possibility that the parasite cytosol contains Hsp70/Hsp40 that facilitate VSG—117 translocation into TbRM was considered. To explore this concept, it was tested whether VSG import into TbRM could be prevented by MAL3-101, a small molecule inhibitor of Hsp40-stimulated Hsp70 ATPase activity that blocks posttranslational import of pre-pro-alpha-factor (ppαMF) into microsomes from S. cerevisiae. Cytosol was pre-incubated with MAL3-101 (or an equal volume of DMSO) while VSG—117 mRNA was translated in a reticulocyte lysate. TbRM was then added to reticulocyte lysate that had been supplemented with cytosol (MAL3-101 or DMSO-treated) containing VSG and the mixture was incubated at 37° C. to promote import of VSG into the microsomes. Each mixture was treated with proteinase K (see FIG. 18A for flow chart of the protocol) in order to detect the imported VSG.
TbRM imported approximately 80% of VSG—117, as measured by protection from proteinase K digestion (FIG. 18B, compare lanes 1, 2), when only DMSO was added to the reaction mixture. In contrast, MAL3-101 inhibited translocation of VSG—117 into ThRM; only 13% of the protein was protected from proteinase K when MAL3-101 was present (FIG. 18B, lanes 3, 4). MAL3-51 (FIG. 5), which is structurally similar to MAL3-101 but has no effect on yeast translocation, had significantly less impact on import of VSG into TbRM even when used at a 3-fold higher concentration (FIG. 18B, lanes 5 and 6). These data indicate that the import of VSG—117 into TbRM is inhibited specifically by MAL3-101.
CJ-21,058 and Equisetin Inhibit Protein Translocation into TbRM. Post-translational protein translocation in E. coli requires SecA whose ATPase activity is inhibited by the small molecule CJ-21,058 (FIG. 5C). In T. brucei, cytosolic chaperones involved in ER protein import have not been completely characterized. The possible involvement of a SecA-like domain in polypeptide import into ThRM was tested using CJ-21,058 to pharmacologically challenge TbRM protein import.
CJ-21,058 inhibited import of VSG—117 into TbRM by 70%, compared to a DMSO control (FIG. 18B, compare lanes 1 and 2 to lanes 9 and 10). Similarly, equisetin, an analog of CJ-21,058 (FIG. 5D) inhibited VSG—117 translocation into TbRM by 95% (FIG. 18B, compare lanes 7 and 8). Thus, a protein with a SecA-like ATPase domain may contribute to the import of VSG—117 into TbRM. Nevertheless, the target of equisetin (and CJ-21,058) in the trypanosome system is not likely to be a protein with extensive sequence similarity to the SecA protein, for two reasons. First, a SECA gene is not encoded in the T. brucei genome. Second, sodium azide, an inhibitor of SecA, does not inhibit translocation of VSG—117 into ThRM. Interesting, SecA contains a DEAD-like domain that is also found in some proteins in T. brucei genome. Therefore, it is possible that equisetin inhibits the activity of a protein with this domain that is required for VSG import into the ER of T. brucei. 
Discussion
Cell-free Protein Import Into Trypanosome ER Microsomes. This example developed a cell-free system using T. brucei microsomes (TbRM) into which trypanosome VSG—117 could be imported post-translationally (FIG. 17B and FIG. 17C) in a signal sequence dependent manner (FIG. 17D). Cytosol from T. brucei is needed for import of full-length VSGs into TbRM (FIG. 17B and FIG. 17C), reminiscent of the requirement for cytosol in the post-translational import of proteins into ER-derived microsomes from the fungi S. cerevisiae and Candida maltosa. 
Factors involved in (or predicted to participate in) the import of proteins into the ER of S. cerevisiae, humans, and T. brucei include: KAR2 (BIP) (in S. cerevisiae, humans, and T. brucei); HSC70 (SSA1) (in S. cerevisiae, humans, and T. brucei); HSP40 (DJ1) (in S. cerevisiae, humans and T. brucei); SEC61α (Sec61p) (in S. cerevisiae, humans, and T. brucei); SEC61β (in S. cerevisiae, and humans); SEC61γ (in S. cerevisiae, humans, and T. brucei); SEC62 (in S. cerevisiae and humans); SEC63 (in S. cerevisiae and humans); SEC71 (in S. cerevisiae); SEC72 (in S. cerevisiae); SRP54 (in S. cerevisiae, humans, and T. brucei); SRP101 (Srα) (in S. cerevisiae, humans, and T. brucei); and SRP102 (Srβ) (in S. cerevisiae and humans). Some of the corresponding genes, for example SEC62, SEC63, SEC71 and SEC72, which are found in S. cerevisiae appear to be absent from the genomes of T. brucei and Archaea. The human genome lacks SEC71 and SEC72, whereas Archaea lack HSC70, a chaperone that is important for post-translational protein translocation in yeasts. However, absence of a protein homolog may preclude activity of a “functional analog” that may provide the needed biochemical function. In fact, for T. brucei there is precedent for this phenomenon; transferrin receptor and the nuclease Dicer lack sequence homology with their mammalian equivalents although the biochemical functions of the proteins are retained. In Archaea, proteins can be secreted post-translationally although the cells appear to lack cytoplasmic Hsp70 as well as Sec71/72p. Functional analogs of Hsp70 (or SecB) probably exist in Archaea. Alternatively, it is possible that the protein secretion pathways evolved independent of these factors.
In T. brucei, three lines of evidence support the existence of post-translational protein translocation into the ER, and suggest that this may be the most common route for entry into the secretory pathway. First, SRP is dispensable for protein import into the secretory pathway, suggesting the existence of an alternative (i.e., chaperone-dependent) route for protein entry into the organelle. Second, T. brucei genome lacks a gene for Srα (SRP102) (Table 1), which is one of the two subunits of the SRP receptor that is required for co-translational protein import into the ER. These facts suggest that the parasite may be unable to efficiently import proteins co-translationally into the ER. The caveat to this view is that a functional analog of SRα exists in T. brucei. Third, we demonstrate posttranslational import of VSG—117 into TbRM in vitro. These data are the most direct evidence that post-translational protein import into the ER can occur in vivo. If SRP is not involved in co-translational import into the ER of the trypanosome, why do the parasites have the genes encoding the complex? SRP has multiple functions in a cell. SRP can direct proteins that lack an N-terminal ER signal sequence to the ER post-translationally. Therefore, one cannot rule out a role for SRP in post-translational protein import into the ER of T. brucei. 
The Secretory Pathway as a Target for Anti-Parasite Drug Discovery. Cell surface proteins, for example VSG, transferrin receptors, nucleobase transporters, and GP63 are important for establishing trypanosomatid infections in humans. Movement of these proteins to the plasma membrane is dependent on entry into the parasite ER, following paradigms worked out in model eukaryotes. The ER is a “gateway” for the transport of proteins to the Golgi, endosomes, lysosomes, and plasma membrane. Small molecules that interfere with import of proteins into the ER of a trypanosome may have value as anti-parasite agents. Moreover, mammals primarily translocate proteins into the ER by the co-translational route, so that compounds that interfere with post-translational ER protein import in T. brucei may compromise viability of the parasite without significant effect on mammalian cells. Data supporting this premise are provided in Example 2.
Molecular chaperones and chaperone-like proteins such as cytoplasmic Hsp70/Hsp40, ER luminal Kar2p/BiP (in eukaryotes), SecB and SecA (in eubacteria) are important for posttranslational protein import. Thus, small molecule modulators of Hsp70/Hsp40 might reduce post-translational import of VSG—117 into TbRM. MAL3-101 inhibits the cochaperone-stimulated ATPase of Hsp70 and inhibits translocation of pre-pro-alpha factor into yeast microsomes. When added to the TbRM protein import system, MAL3-101 (FIG. 5A) inhibited translocation of VSG—117 into parasite microsomes (FIG. 18B, lanes 3 and 4). Further, MAL3-101 killed T. brucei (Example 2). The efficacy of MAL3-101 against T. brucei (IC50 was 1.5 μM) is comparable to that of suramin, a drug that is used to treat human African trypanosomiasis (IC50 for suramin is 1.4-2.3 μM). These data indicate that MAL3-101 is a chemical entity that is worthy of optimization for anti-trypanosome drug discovery.
Equisetin (FIG. 5D) and CJ-21,058 (FIG. 5C), both fungal products inhibited import of VSG—117 into TbRM (FIG. 18), and the compounds were trypanocidal (Example 2). Fortunately, a total synthesis of equisetin has been described (Burke et al, 2000, Org Lett; 2:3611-3), an achievement that could guide future efforts, in the light of their anti-trypanosomal effects, to synthesize bioactive analogs for structure-activity studies on T. brucei. Finally, it will be interesting to test whether other equisetin-related compounds (e.g., altersetin, hexahydroaltersetin, dihydroaltersetin, tetrahydroequisetin, and phomasetin) (De Clercq, 2000, Med Res Rev; 20:323-49; Hellwig et al., 2002, J Antibiot (Tokyo); 55:881-92; Tziveleka, 2003, Curr Top Med Chem; 3:1512-35) are trypanocidal.
Example 11
VSG Binding to T. brucei Microsomes (TbRM)
Proteins imported into the ER must first bind to the membrane of the organelle before translocation across the lipid bilayer. Further, the imported proteins remain associated with intact microsomal vesicles after their import into the ER. This example demonstrates an VSG—117 association with TbRM, an important step in the translocation pathway. In a “membrane flotation” study without TbRM, 90% of VSG—117 was detected in the 2.3 M sucrose layer, with only 5% in the 1.5 M sucrose layer where membranes sediment (FIG. 19C lanes 2 and 3). When TbRM was added to the mixture, 4-fold more VSG (i.e., 20%) was membrane-bound, in the 1.5 M sucrose cushion (FIG. 19D). This data is consistent with our earlier conclusions; VSG—117 binds to TbRM and is imported into the microsomes (Example 10).
Specifically, VSG-117 mRNA was translated in rabbit reticulocyte lysate for one hour at 37° C. Cycloheximide was added to stop protein synthesis. Two 5 μl aliquots of reaction mixture were aliquoted to new tubes. One aliquot was left untreated while 1 μl of TbRM was added to the other aliquot. The samples were incubated at 37° C. for 1.5 hours. Translocation was stopped by incubating the samples on ice. As shown in FIG. 19A, carbonate extraction of VSG-117 in the absence or presence of TbRM was performed using similar methods to those described in Zhang et al. (Zhang et al., 2001, Mol Biol Cell; 12(5):1303-14). Sodium carbonate (100 mM) was added to the samples and incubated on ice for 30 minutes. Next, the samples were centrifuged at 230,000×g for one hour. The supernatant from each sample was extracted, precipitated with ammonium sulfate for 30 minutes on ice, and pelleted at 16,060×g for 10 minutes at 4° C. The pellets from the 230,000×g and 16,060×g centrifugation steps were resuspended in SDS sample buffer, resolved by Tris-Tricine SDS-PAGE, and quantitated using a phosphorimager and Quantity One software. In FIG. 19A, lanes 1 and 2 represent VSG-117 carbonate extraction without TbRM. Lanes 3 and 4 represent VSG-117 carbonate extraction with TbRM present. “S” denotes proteins precipitated from the supernatant of the 230,000×g step while “P” represents proteins recovered from the 230,000×g centrifugation step. Brackets denote lanes that were compared to obtain a percentage to total VSG-117 present. FIG. 19B presents the quantitation of data from FIG. 19A. Percentage of total VSG-117 present is expressed as the amount of VSG detected in a single lane divided by the sum of the amount of VSG detected in the supernatant and pellet lanes.
In FIG. 19C the floatation of VSG-117 in the absence (top panel) or presence (bottom panel) of TbRM was performed, again using similar methods to those described by Zhang et al. [35S]Met-labelled VSG117 was translated in a rabbit reticulocyte lysate and incubated with or without TbRM. Reaction mixtures were combined with 2.3 M sucrose in rough microsome buffer (RMB) (2.3 M Top) and layered onto a bottom layer of 2.3 M sucrose in RMB (2.3 M Bottom). Next, RMB containing 1.5 M sucrose was layered above the 2.3M layer, and RMB containing 0.25 M sucrose was layered above the 1.5 M layer. The sucrose gradient was then centrifuged at 100,000×g for five hours at 4° C. Each layer was then extracted, precipitated with ammonium sulfate, resuspended in SDS sample buffer, resolved using Tris-Tricine SDS-PAGE, and quantitated using a phosphorimager and Quantity One software. In FIG. 19C, lane 1 represents proteins obtained from the 0.25 M sucrose layer; lane 2 represents proteins obtained from the 1.5 M sucrose layer; lane 3 represents proteins obtained from the top 2.3 M sucrose layer; and lane 4 represents proteins recovered from the bottom 2.3 M sucrose layer and those protein pelleted from the 100,000×g centrifugation step. FIG. 19D presents a quantitation of data from FIG. 19C. The percentage of total VSG-117 present is expressed as the amount of VSG detected in a single lane divided by the sum of VSG detected in lanes 1-4. “*” Represents 0% VSG-117 detected.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
Sequence Listing Free Text


    SEQ ID NO: 1-4, 22-26 synthetic oligonucleotide primers
    SEQ ID NO:5-13, 17-21 amino acid sequences of VSG-117h constructs
    SEQ ID NO:14-16 amino acid sequences of h-motifs

## Claims

1. A composition comprising:
as one component an isolated cell free preparation of protozoan microsomes; the cell free preparation of protozoan microsomes the resuspended pellet of a low speed spin of about 5,000 g to about 20,000 g of a lysate of a protozoan of the genus Trypanosoma selected from the group consisting of T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense; and translocates a polypeptide into the microsome;
and as a second component an isolated purified cell free preparation of protozoan cytosol; the preparation of protozoan cytosol the supernatant of a high speed spin of greater than 20,000 g of a lysate of a protozoan of the genus Trypanosoma selected from the group consisting of T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense; and facilitates the translocation of a plasma membrane polypeptide into a cell free preparation of protozoan microsomes.

2. The composition of claim 1 wherein the translocated polypeptide is protected from exogenous detergent.

3. The composition of claim 1, wherein the composition facilitates the translocation of a VSG 117500 substrate into the cell free preparation of protozoan microsomes.

4. A composition comprising:
as one component an isolated cell free preparation of protozoan microsomes; the cell free preparation of protozoan microsomes the resuspended pellet of a low speed spin of about 5,000 g to about 20,000 g of a lysate of a protozoan of the genus Trypanosoma; and translocates a polypeptide into the microsome;
and as a second component an isolated purified cell free preparation of protozoan cytosol; the preparation of protozoan cytosol the supernatant of a high speed spin of greater than 20,000 g of a lysate of a protozoan of the genus Trypanosoma; and facilitates the translocation of a plasma membrane polypeptide into a cell free preparation of protozoan microsomes.

5. The composition of claim 4, wherein the composition facilitates the translocation of a VSG 117500 substrate into the cell free preparation of protozoan microsomes.

6. The composition of claim 4 wherein the translocated polypeptide is protected from exogenous detergent.

7. A method of endoplasmic reticulum (ER) translocation of a polypeptide; the method comprising contacting a polypeptide comprising a translocation signal sequence of protozoan origin with a composition of claim 1.

8. A method of screening for an agent that modulates the ER translocation of a polypeptide in a protozoan, the method comprising:
contacting the composition of claim 1 with an agent;
and monitoring ER translocation of a polypeptide comprising a translocation signal sequence of protozoan origin;
wherein a modulation in the ER translocation of the polypeptide indicates the agent modulates ER translocation of a polypeptide in a protozoan.

9. A method of screening for an agent for the treatment and/or prevention of a protozoan infection, the method comprising: contacting the composition of claim 1 with an agent; and monitoring ER translocation of a polypeptide comprising a translocation signal sequence of protozoan origin; wherein a modulation in the ER translocation of the polypeptide indicates the agent is a candidate for the treatment and/or prevention of a protozoan infection.

10. The method of claim 9, wherein the modulation is a decrease or inhibition in the ER translocation of the polypeptide.

11. A method of screening for an agent that kills, inhibits the growth, and/or inhibits the reproduction of a protozoan, the method comprising:
contacting the composition of claim 1 with an agent;
and monitoring ER translocation of a polypeptide comprising a translocation signal sequence of protozoan origin;
wherein a modulation in the ER translocation of the polypeptide indicates the agent is a candidate agent that kills, inhibits the growth, and/or inhibits the reproduction of a protozoan.

12. The method of claim 11, wherein the modulation is a decrease or inhibition in the ER translocation of the polypeptide.

13. The method of claim 7 wherein the translocated polypeptide is a soluble secreted polypeptide or a plasma membrane polypeptide.

14. The method of claim 7 wherein the translocated polypeptide is a polypeptide of nonprotozoan origin with a protozoan signal sequence.

15. The method of claim 8 wherein the translocated polypeptide is protected from exogenous detergent.

16. The method of claim 9 wherein the translocated polypeptide is protected from exogenous detergent.

17. The method of claim 11 wherein the translocated polypeptide is protected from exogenous detergent.