bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence requirements for stabilization of a peptide reverse turn in water solution
Authors: Dyson H. Jane , Bolinger Lizann , Feher Victoria A. , Osterhout Jr John J. , Yao Jian , Wright Peter E.
Published: March 2003

To probe the sequence requirements for stabilization of a reverse turn conformation in a short peptide in water solution, the behavior of two series of peptides was investigated by nuclear magnetic resonance (NMR) spectroscopy. The peptides have the general sequences XPGDV and AXGDV, where X is a representative subset of all 20 naturally occurring amino acids. The residues chosen at positions 3 and 4, Gly and Asp, respectively, were shown to give the greatest population of reverse turns in a previous study [Dyson, H. J., Rance, M., Houghten, R. A., Lerner, R. A. & Wright, P. E. (1988) J. Mol. Biol. 201, 161−200]. Within this framework, the identity of the first residue of the turn (X in XPGDV) does not greatly influence the turn population, although a small but significant increase is observed for residues such as Ala which have a preference for backbone conformations in the α region of (φ,ψ) space. The series AXGDV was initially studied for completeness only, since it was expected that the turn would not be stabilized in such a small linear peptide in the absence of proline. In contrast, it appears that a significant population of type II turn conformations is to be found in peptides in the series AXGDV, although proline remains one of the most favorable residues at position 2. These results indicate that while residues at all positions within the turn can influence the turn population, the presence of Gly‐Asp as the third and fourth members of the sequence gives a strong bias towards type II turn formation regardless of the residues at positions 1 and 2. Our results give a final prediction that the sequence with the highest intrinsic propensity for turn formation is APGD.
Myxococcus xanthus spore coat protein S, a stress‐induced member of the βγ‐crystallin superfamily, gains stability from binding of calcium ions
Authors: Wenk Martina , Mayr Eva‐Maria
Published: March 2003

Protein S, a calcium‐binding spore coat protein from the soil bacterium Myxococcus xanthus, belongs to a group of structurally related proteins, the βγ‐crystallin superfamily. Common features of this protein family are the Greek‐key structural motif or crystallin fold, and the fact that all members are extremely stable long term. To investigate the correlation between the stability and Greek‐key topology, protein S was cloned, expressed in Escherichia coli and purified to homogeneity. Ca2+ binding influences the native tertiary structure of protein S, whereas the secondary structure remains unaffected as shown by spectroscopic methods. Ca2+ ions enhance the conformational stability of protein S significantly. The midpoints of urea and guanidinium chloride‐induced transitions show a difference of 1.4 M and 0.5 M denaturant, respectively, in the absence and in the presence of calcium. An equilibrium intermediate indicating independent domain folding can be detected at pH 2. In addition, thermal denaturation shows a clear deviation from the two‐state model of folding, again with a strong stabilisation by Ca2+ ions. Temperature and denaturant‐induced equilibrium transitions are fully reversible. Our data implicate a different strategy for achieving the high stability required for the biological function compared with the structurally related lens crystallins.
Cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA hydratase, an enzyme involved in anaerobic metabolism of benzoyl‐CoA in the denitrifying bacterium Thauera aromatica
Authors: Laempe Diana , Eisenreich Wolfgang , Bacher Adelbert , Fuchs Georg
Published: March 2003

Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl‐CoA as the common intermediate. The central pathway of benzoyl‐CoA metabolism is initiated by an ATP‐driven reduction of the aromatic ring producing cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA. The 1,5‐dienoyl‐CoA intermediate is thought to be transformed to 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA by a specific dienoyl‐CoA hydratase catalyzing the formal addition of water to one of the double bonds.

This dienoyl‐CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica and was found to be highly specific for the cyclic 1,5‐dienoyl‐CoA. A second 29‐kDa enoyl‐CoA hydratase acted on crotonyl‐CoA; this highly active enoyl‐CoA hydratase also acted slowly on cyclohex‐1‐ene‐1‐carboxyl‐CoA. The regulation of expression of dienoyl‐CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl‐CoA hydratase is the second enzyme of the central benzoyl‐CoA pathway of anaerobic aromatic metabolism in T. aromatica.

Extracts of Rhodopseudomonas palustris contained high activity of cyclohex‐1‐ene‐1‐carboxyl‐CoA hydratase, but no 1,5‐dienoyl‐CoA hydratase activity. It appears that a variant of the benzoyl‐CoA pathway is operating in R. palustris in which hydration of the 1,5‐dienoyl‐CoA does not take place. Rather, cyclohex‐1‐ene‐1‐carboxyl‐CoA is hydrated to 2‐hydroxycyclohexane‐1‐carboxyl‐CoA.
Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis
Authors: Krafft Torsten , Macy Joan M.
Published: March 2003

Chrysiogenes arsenatis is the only bacterium known that respires anaerobically using arsenate as the terminal electron acceptor and the respiratory substrate acetate as the electron donor. During growth, the arsenate is reduced to arsenite; the reduction is catalyzed by an arsenate reductase. This study describes the purification and characterization of a respiratory arsenate reductase (Arr). The enzyme consists of two subunits with molecular masses of 87 kDa (ArrA) and 29 kDa (ArrB), and is a heterodimer α1β1 with a native molecular mass of 123 kDa. The arsenate reductase contains molybdenum, iron, acid‐labile sulfur and zinc as cofactor constituents. The Km of the enzyme for arsenate is 0.3 mM and the Vmax is 7013 μmol arsenate reduced  min−1  mg protein−1. Nitrate, sulfate, selenate and fumarate cannot serve as alternative electron acceptors for the arsenate reductase. Synthesis of the protein is regulated, as arsenate must be present during growth for the enzyme to be fully induced. The N‐terminus of ArrA is similar to a number of procaryotic molybdenum‐containing polypeptides (e.g. the formate dehydrogenases H and N of Escherichia coli). The N‐terminus of ArrB is similar to iron‐sulfur proteins. The respiratory arsenate reductase of C. arsenatis is different from the non‐respiratory arsenate reductases of E. coli and Staphylococcus aureus.
Structural elucidation of the lipopolysaccharide core regions of the wild‐type strain PAO1 and O‐chain‐deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5
Authors: Sadovskaya Irina , Brisson Jean‐Robert , Lam Joseph S. , Richards James C. , Altman Eleonora
Published: March 2003

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild‐type strain PAO1 and derived rough‐type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum‐ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of
D‐glucose (four units),
L‐rhamnose (one unit), 2‐amino‐2‐deoxy‐
D‐galactose (one unit),
Lglycero‐
D‐manno‐heptose (Hep; two units), 3‐deoxy‐
D‐manno‐octulosonic acid (Kdo; two units),
Lalanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one‐dimensional and two‐dimensional (2D) NMR techniques in combination with MS‐based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two‐dimensional 31P (ω1)‐half‐filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild‐type strain. The presence of a 7‐O‐carbamoyl substituent was observed on the second heptose. The structure of the core region of the O‐chain‐deficient LPS from P. aeruginosa serotype O5 is as follows :
R2
CONH2P↓↓↓
3
7
6
R1←4)‐α‐
D‐GalpN‐(1←3)‐
L‐α‐
D‐Hepp‐(1←3)‐
L‐α‐
D‐Hepp‐(1←5)‐α‐
D‐Kdop‐(2←6)‐lipid A
2
2
4
4
↑↗↖↑L‐Ala
P
P
2
α‐
D‐Kdop
where R1 is β‐
D‐Glcp‐(1←2)‐α‐
L‐Rhap‐(1←6)‐α‐
D‐Glcp‐(1← and R2 is α‐
D‐Glcp‐(1←6)‐β‐
D‐Glcp‐(1←.

A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.
Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the γ‐cyclodextrin production
Authors: Parsiegla Goetz , Schmidt Andreas K. , Schulz Georg E.
Published: March 2003

Bacterial cyclodextrin glycosyltransferases use starch to produce cyclic maltooligosaccharides (cyclodextrins) which are of interest in various applications. The cyclization reaction gives rise to a spectrum of ring sizes consisting of predominantly six to eight glucosyl units. Using the enzyme from Bacillus circulans strain no. 8, binding studies have been performed with several substrates and analogues. The observed binding modes differ in detail, but agree in general with data on homologous enzymes. Based on these binding studies, two mutations were designed that changed the production spectrum from the predominant product β‐cyclodextrin of the wild‐type enzyme towards γ‐cyclodextrin, which is of practical interest because it is rare and can encapsulate larger nonpolar compounds.
Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors
Authors: Frolova Lyudmila Y. , Simonsen Janne L. , Merkulova Tatyana I. , Litvinov Dmitry Y. , Martensen Pia M. , Rechinsky Vladimir O. , Camonis Jacques H. , Kisselev Lev L. , Justesen Just
Published: March 2003

Translation termination in eukaryotes is governed by termination codons in mRNA and two release factors, eRF1 and eRF3. In this work, human eRF1 and eRF3 have been produced in insect cells using a recombinant baculovirus expression system for the corresponding human cDNAs. Purification of eRF1 has led to a homogeneous 50‐kDa protein active in promoting ribosome‐dependent and termination‐codon‐dependent hydrolysis of formylmethionyl‐tRNA.esMet.rb.eif.rb. Purification of eRF3 yielded a full‐length protein and shorter polypeptides. Microsequencing of the N‐terminus of the shortest form detected a site of proteolytic cleavage between Arg91 and Gly92, probably due to exposed region(s) hypersensitive to proteolysis. The mixture of full‐length and truncated forms of eRF3 as well as bacterially expressed eRF3 lacking 138 N‐terminal amino acids (eRF3Cp) are active as an eRF1‐dependent and ribosome‐dependent GTPase and in stimulating the GTP‐dependent release activity of eRF1. Complex formation between eRF1 and eRF3Cp was demonstrated by affinity and gel‐filtration chromatographies and by native‐gel electrophoresis. An abnormal electrophoretic mobility observed for eRF1 as compared with the complex points to a significant conformational change of either eRF1 or both factors in the complex. Co‐expression of both factors in baculovirus‐infected insect cells and a yeast two‐hybrid assay were applied to monitor complex formation in vivo. In yeast cells, both eRF1 and eRF3 are either in a monomeric or in a heterodimeric but not in a homodimeric state.
Changes in myosin mRNA and protein expression in denervated rat soleus and tibialis anterior
Authors: Huey Kimberly A. , Bodine Sue C.
Published: March 2003

Denervation differs from other models of reduced neuromuscular activation due to the absence of a nerve‐muscle connection and limited data exists regarding the effects of denervation on myosin heavy chain (MHC) expression. Thus, adult MHC expression (I, IIa, IIx, IIb) was studied in the rat soleus and tibialis anterior (TA) at the mRNA and protein levels 2, 4, 7, 10, 14, and 30 days following sciatic nerve transection. MHC protein content was quantified with SDS/PAGE and mRNA levels with the RNase‐protection assay. Control soleus consisted predominately of type I MHC mRNA and protein, however, 4 days after denervation type I MHC mRNA was significantly decreased to 41 ± 8 % of control and continued to remain below control values. Soleus IIa mRNA was significantly elevated 7 and 10 days after denervation while IIx mRNA remained relatively constant until 30 days when it increased to 197 ± 23 % of control. At the protein level, soleus I MHC significantly decreased to 80 % of the total while IIa MHC significantly increased to 20 % of the total. At 30 days, IIx MHC protein accounted for 9.4 ± 1.6 % of the total soleus MHC protein. In the TA, IIb mRNA was significantly decreased to 57 % of control by day 4 and remained significantly decreased for up to a month. TA IIx mRNA was also significantly decreased at 10 and 30 days after denervation. Similar to the soleus, TA IIa mRNA was significantly increased over control 7−14 days after denervation. There were no significant changes in TA MHC protein profile during one month of denervation. In both the soleus and TA, denervation significantly shifted the MHC mRNA profile as early as 4 days following denervation without any corresponding changes at the protein level. Significant mRNA changes without large changes in MHC protein composition continued throughout the denervation period suggesting that the muscle may be prevented from premature functional transitions by mechanisms such as decreased mRNA stability, translational block, or increased turnover of newly synthesized proteins.
Solution structures of the melanocyte‐stimulating hormones by two‐dimensional NMR spectroscopy and dynamical simulated‐annealing calculations
Authors: Lee Jung‐Hoon , Lim Sung‐Kil , Huh Sung‐Ho , Lee Donghan , Lee Weontae
Published: March 2003

Melanocortins, which are involved in melanocyte pigmentation control and glucocorticoid stimulation, have functional roles in various physiological mechanisms and have been shown to participate in higher cortical functions. Recently, it has also been reported that melanocyte‐stimulating hormone (MSH) and melanocortin 4 receptor (MC4R) are the key components of the hypothalamic response to obesity. The solution structures of both melanocyte‐stimulating hormone α‐MSH (Ac‐Ser‐Tyr‐Ser‐Met‐Glu‐His‐Phe‐Arg‐Trp‐Gly‐Lys‐Pro‐Val‐NH2) and its analog α‐MSH‐ND (Ac‐Ahx‐Asp‐His‐
DPhe‐Arg‐Trp‐Lys‐NH2) (Ahx, 2‐aminohexanoic acid) have been determined by two‐dimensional NMR spectroscopy and simulated‐annealing calculations. The NMR data revealed that α‐MSH forms a hairpin loop conformation which includes conserved message sequences, whereas α‐MSH‐ND prefers a type I β‐turn comprising residues of Asp2‐His3‐
DPhe4‐Arg5. Final simulated‐annealing structures of both α‐MSH‐ND and α‐MSH peptides converged with rmsd of 0.07 nm for α‐MSH‐ND and 0.1 nm for α‐MSH between backbone atoms, respectively. This result will provide the structural bases of melanocortin functions as well as valuable information for structure‐based drug design involving the regulation of obesity and feeding.
The lactose transporter of Staphylococcus aureus
Authors: Kowolik Claudia M. , Hengstenberg Wolfgang
Published: March 2003

The lactose‐specific enzyme II (IICBlac) of the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) of Staphylococcus aureus couples translocation to phosphorylation of the transported lactose. It is composed of the N‐teminal membrane‐bound IIC domain, which includes the sugar‐binding site, and the C‐terminal IIB domain, which contains the phosphorylation site at Cys476. IIC (residues 1−461) fused with a C‐terminal affinity tag of six histidine residues and IIB (residues 461−570) fused with an N‐terminal histidine tag were overexpressed in Escherichia coli and purified by Ni2+ chelate affinity chromatography. 2 mg of IIClac‐His6 obtained from 10 g of cells and 12 mg of His6‐IIBlac obtained from 8 g of wet cells were purified to homogeneity. 56 % of the total IIClac‐His6 activity present in the membranes could be recovered. Purification by affinity chromatography yields the opportunity to exchange the detergent. The Km determined in an activity assay for IIClac‐His6 in the presence of the histidine‐tagged IIBlac domain (His6‐IIBlac) was similar to the Km determined for histidine‐tagged IICBlac‐His [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798−804], suggesting that substrate affinity is barely influenced by the expression of the domains as separate proteins. The Vmax is reduced by a factor of 25 compared with IICBlac‐His. His6‐IIBlac also complements the activity of the IICBlac mutant C476S, which possesses an inactive IIB domain. This result indicates that IIC and IIB are flexibly linked in such a way that free His6‐IIBlac can displace the inactive IIB domain from its contact site on the IIC domain. His6‐IIBlac is shorter and more stable than a previously constructed IIB domain (IIBlac‐His) [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798−804)], which contained a C‐terminal histidine tag. The Km values for phosphoenolpyruvate‐dependent phosphorylation of His6‐IIBlac and IIBlac‐His are nearly indistinguishable, suggesting that the location of the affinity tag either at the N‐terminal or at the C‐terminal end of the domain does not influence the substrate affinity.
Stimulation of fructose 1,6‐bisphosphate production in melanoma cells by α‐melanocyte‐stimulating hormone
Authors: Tyagi Rakesh Kumar , Azrad Anat , Degani Hadassa , Salomon Yoram
Published: March 2003

In a 31P‐NMR spectroscopic study of cultured M2R mouse melanoma cells, we previously demonstrated the acute stimulation of three peaks in the phosphomonoester region of the spectrum by [Ahx4,
DPhe7]α‐melanotropin (concomitant with an increase in cellular adenosine 3′,5′‐phosphate (cAMP) and a decrease in ATP [Degani, H., DeJordy, J. O. & Salomon, Y. (1991) Proc. Natl Acad. Sci. USA 88, 1506−1510]. Chemical identification of these metabolites was performed in this study using 32P metabolic labeling and polyethyleneimine‐cellulose thin layer chromatography in combination with 31P‐NMR and 13C‐NMR spectroscopic methods. Two of the stimulated signals were identified as P1 and P6 of fructose 1,6‐bisphosphate (FruP2) and their mode of regulation by α‐melanotropin was examined. The FruP2 response to α‐melanotropin coincided in time and dose with a rise in cAMP and a decrease in levels of ATP, while elevation of cAMP by forskolin alone did not increase FruP2. The stimulatory effect of αmelanotropin was not associated with a change in the overall rate of glycolysis, suggesting that FruP2 levels were not rate limiting in this process. The data suggest the presence of a previously unknown response of M2R melanoma cells to α‐melanotropin, which coincides in time with enhanced cAMP accumulation but is not mediated by cAMP and may relate to the control of FruP2 in a non glycolytic context.
Transcriptional regulation of the tissue‐type plasminogen‐activator gene in human endothelial cells : identification of nuclear factors that recognise functional elements in the tissue‐type plasminogen‐activator gene promoter
Authors: Costa Magdaline , Shen Yang , Maurer Fabienne , Medcalf Robert L.
Published: March 2003

The gene encoding human tissue‐type plasminogen activator (t‐PA) is regulated in a cell‐type‐specific manner. Previous studies in non‐endothelial cells have indicated that basal and phorbol ester mediated induction is controlled by a cAMP response element (CRE) referred to as the tPACRE, and an activating protein 2 (AP‐2)‐like site. The classification of the AP‐2‐like site was assigned on the basis of its sequence homology, but has been shown in some cell systems to be recognised by promoter‐specific transcription factor‐1 (Sp‐1). Here, we have investigated the transcriptional regulation of the t‐PA gene in endothelial cells and addressed the functional roles of the tPACRE and the Sp‐1/AP‐2‐like sites. 5′‐RACE experiments indicate that the t‐PA gene uses two transcription initiation sites in these cells with the downstream site being preferred. Functional analyses of the t‐PA promoter using reporter‐gene constructs transfected into C11STH endothelial cells demonstrate that the first 410 bp of the t‐PA promoter confers an increase in reporter‐gene activity on treatment with 4β‐phorbol 12‐myristate 13‐acetate (PMA). Mutagenesis of either the tPACRE or the Sp‐1/AP‐2 site weakens both basal and inducible expression, while disruption of both sites renders the promoter completely unresponsive. Using supershift assays, we identify the predominant tPACRE‐binding proteins in nuclear extracts prepared from both C11STH cells and primary umbilical vein endothelial cells (HUVECs) as activating transcription factor 2, CREB (cAMP‐responsive‐element‐binding protein), CREM (cAMP response element modulator) and c‐jun. Treatment of cells with PMA results in a selective recruitment of jun‐D to the tPACRE, while Sp‐1 was identified as the major transcription factor that recognises the AP‐2‐like site. Based on this data and previous reports, we have reassigned this as a Sp‐1‐binding site. Finally, the identification of specific endothelial‐derived t‐PACRE‐binding proteins suggests an integral role for these factors in the regulation of t‐PA gene expression in human endothelial cells.
Transducin‐like Enhancer of split 2, a mammalian homologue of Drosophila Groucho, acts as a transcriptional repressor, interacts with Hairy/Enhancer of split proteins, and is expressed during neuronal development
Authors: Grbavec Diane , Lo Rita , Liu Yanling , Stifani Stefano
Published: March 2003

Groucho is a Drosophila transcriptional repressor involved in neurogenesis, segmentation, and sex determination together with basic helix‐loop‐helix proteins of the Hairy/Enhancer of split (HES) family. Several mammalian Groucho homologues, the Transducin‐like Enhancer of split (TLE) 1 through 4 proteins, share similar properties with their Drosophila counterpart, suggesting that TLE proteins perform functions analogous to the roles of Groucho in Drosophila. The aim of this study was to examine this possibility by characterizing the properties of TLE2 and extending the analysis of TLE1. It is shown here that TLE2 and TLE1 are transcriptional repressors that contain two separate repression domains, located either within a Gln‐rich amino terminal region or within an internal domain characterized by an abundance of Ser, Thr, and Pro residues. In addition, both TLE2 and TLE1 can homo‐ and heterodimerize through a short region that is part of their amino‐terminal transcription repression domains. Finally, TLE2 interacts and is co‐expressed with mammalian HES proteins in both neural and non‐neural tissues. Taken together, these findings implicate TLE2 in transcriptional repression and define the structural elements that mediate transcriptional and protein−protein interaction functions of Groucho/TLE proteins.
Transport of arginine and ornithine into isolated mitochondria of Saccharomyces cerevisiae
Authors: Soetens Oriane , Crabeel Marjolaine , El Moualij Benaïssa , Duyckaerts Claire , Sluse Francis
Published: March 2003

In this work we have characterised the transport of L‐arginine and
L‐ornithine into mitochondria isolated from a wild‐type Saccharomyces cerevisiae strain and an isogenic arg11 knock‐out mutant. The Arg11 protein (Arg11p) is a mitochondrial carrier required for arginine biosynthesis [Crabeel, M., Soetens, O., De Rijcke, M., Pratiwi, R. & Pankiewicz, R. (1996) J. Biol. Chem. 271, 25 011−25 019]. Reconstitution experiments have confirmed that it is an
L‐ornithine carrier also transporting
L‐arginine and
L‐lysine by order of decreasing affinity, but not
L‐histidine [Palmieri, L., De Marco, V., Iacobazzi, V., Palmieri, F., Runswick, M. & Walker, J. (1997) FEBS Lett. 410, 447−451]. Evidence is presented here that the mitochondrial inner membrane contains an
L‐arginine and
L‐ornithine transporting system distinct from Arg11p, in keeping with the arginine leaky phenotype of arg11 knock‐out mutants. The newly characterised carrier, which we propose to name Bac1p (basic amino acid carrier), behaves as an antiporter catalysing the electroneutral exchange of the basic amino acids
L‐arginine,
L‐lysine,
L‐ornithine and
L‐histidine and displays the highest affinity for
L‐arginine (Km of 30 μM).
L‐Arginine uptake has a pH optimum in the range of 7.5−9 and is inhibited by several sulphydryl reagents, by pyridoxal 5′‐phosphate and by cations.
Protein LA, a novel hybrid protein with unique single‐chain Fv antibody‐ and Fab‐binding properties
Authors: Svensson Henrik G. , Hoogenboom Hennie R. , Sjöbring Ulf
Published: March 2003

Existing Ig‐binding proteins all suffer from limitations in their binding spectrum. In the pursuit of the ultimate, non‐restricted, Ig‐binding protein, we have constructed the hybrid protein LA, by fusing four of the Igκ light‐chain‐binding domains of peptostreptococcal protein L with four of the IgGFc‐ and Fab‐binding regions of staphylococcal protein A. Ligand‐blot experiments demonstrated that the L and the A components were both functional in the hybrid, as the protein was shown to bind purified κ light chains and IgGFc. Protein LA bound human Ig of different classes and IgG from a wide range of mammalian species. IgG, IgM and IgA were purified from human serum and saliva by affinity chromatography on protein LA agarose. Similarly, single‐chain Fv (scFv) antibodies carrying the κ light‐chain variable domain or expressing the VHIII (variable domain of the heavy chain of Ig) determinant, were efficiently purified on immobilized protein LA. As judged by surface plasmon resonance (SPR), protein LA showed enhanced affinity for all tested ligands, including several scFv antibodies, compared with proteins L and A alone. SPR analysis also demonstrated that binding of a ligand to one of the components in protein LA did not affect the ability of the hybrid protein to interact simultaneously with a ligand for the other component. The antigen‐binding capacity of a κ‐expressing scFv antibody was unaffected by the interaction with protein LA, whereas the binding of a VHIII‐expressing scFv antibody to its antigen was, unexpectedly, blocked by protein A and protein LA. Together, these data demonstrate that protein LA represents a highly versatile Ig‐binding molecule.
The mammalian small heat‐shock protein Hsp20 forms dimers and is a poor chaperone
Authors: van de Klundert Francy A. J. M. , Smulders Ronald H. P. H. , Gijsen Mariken L. J. , Lindner Robyn A. , Jaenicke Rainer , Carver John A. , de Jong Wilfried W.
Published: March 2003

Hsp20 is one of the newly described members of the mammalian small heat‐shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far‐ultraviolet circular dichroism spectra as the most closely related sHsp, αB‐crystallin, but was less heat stable, denaturing upon heating to 50 +C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43‐kDa dimers and 470‐kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone‐like activity than αB‐crystallin, as indicated by its relatively poor capacity to diminish the reduction‐induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C‐terminus and less polar than other sHsps, but 1H‐NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C‐terminal extension, may contribute to the less effective chaperone‐like activity.
Characterization of human T‐cell leukemia virus type I integrase expressed in Escherichia coli
Authors: Müller Barbara , Kräusslich Hans‐Georg
Published: March 2003

The C‐terminal part of the pol gene of the human T‐cell leukemia virus type I (HTLV‐I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV‐I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV‐I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR‐Pol precursor by proteolytic cleavage. HTLV‐I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size‐exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5‐derived vs. U3‐derived sequences in the processing and strand‐transfer reactions. In the disintegration reaction, HTLV‐I IN not only accepted the double‐stranded branched substrate corresponding to the product of a strand‐transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single‐stranded or a single adenosine overhang.
Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion
Authors: Lang Jochen
Published: March 2003

Secretion of the peptide hormone insulin from pancreatic β cells constitutes an important step in the regulation of body homeostasis. Insulin is stored in large dense core vesicles and released by exocytosis, a multistage process involving transport of vesicles to the plasma membrane, their docking, priming and finally their fusion with the plasma membrane. Some of the protein components necessary for this process have been identified in β cells. The export of potent and potentially harmful substances has to be tightly controlled. The secretory response in pancreatic β cells requires the concerted action of nutrients together with enteric hormones and neurotransmitters acting on G‐protein coupled receptors. It is well established that glucose and other metabolizable nutrients depolarize the β‐cell membrane and the ensuing Ca2+ influx through voltage‐dependent channels constitutes a main stimulus for insulin exocytosis. Theoretical considerations and recent observations suggest in addition an organizing role for the Ca2+ channel similar to neurotransmission. A second regulatory control on exocytosis is exerted by monomeric and heterotrimeric G‐proteins. The monomeric GTPase Rab3A controls insulin secretion through cycling between a guanosine triphosphate liganded vesicle‐bound form and a guanosine diphosphate liganded, cytosolic form. The effect of neurohormones is transduced by the heterotrimeric GTPases. Whereas pertussis‐toxin sensitive α‐subunits exert direct inhibition at the level of exocytosis, the Gβγ‐subunits are required for stimulation. It is possible that these GTPases exert immediate regulation, while protein kinases and phosphatases may modulate long‐term adaptation at the exocytotic machinery itself. The molecular nature of their activators and effectors still await identification. Insights into the progression of the exocytotic vesicle from docking to fusion and how these processes are precisely regulated by proteins and second messengers may provide the basis for new therapeutic principles.
Structure of the human β2‐glycoprotein I (apolipoprotein H) gene*
Authors: Okkels Henrik , Rasmussen Thomas E. , Sanghera Dharambir K. , Kamboh M. Ilyas , Kristensen Torsten
Published: March 2003

The gene encoding the human plasma protein β2‐glycoprotein I or apolipoprotein H was cloned and its structure determined. The gene which consists of eight exons was shown to span 18 kb and was localized to chromosome 17q23‐24. The transcriptional initiation site was assigned to a position 31 bp upstream of the start codon. Several consensus sequence elements relevant for regulation of transcription in liver were seen in the 5′‐upstream region of the gene. Exon 1 contains the 5′‐UTR together with the signal peptide coding sequences. Short consensus repeats (SCRs) 1, 3, 4, and 5 are encoded by single exons each while SCR2 is encoded by two exons. Exon 8 comprises the region encoding the C‐terminal end of β2‐glycoprotein I (from His‐310), the stop codon and the 3′‐UTR.
Structural organization and expression of the mouse gene for Pur‐1,  a highly conserved homolog of the human MAZ gene
Authors: Song Jun , Murakami Hiroo , Tsutsui Hatsumi , Ugai Hideyo , Geltinger Christian , Murata Takehide , Matsumura Masatoshi , Itakura Keiichi , Kanazawa Ichirou , Sun Kailai , Yokoyama Kazunari K.
Published: March 2003

We have characterized the genomic structure and expression of the mouse gene for Pur‐1. The cloned Pur‐1 gene spans a 5‐kb region encompassing the promoter, five exons, four introns and the 3′‐untranslated region. All exon–intron junction sequences conform to the GT/AG rule. The promoter region has typical features of a housekeeping gene: a high G + C content (77.5%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 108 nucleotides upstream of the ATG codon. Comparison of Pur‐1 with the human gene for MAZ (Myc‐associated zinc finger protein) revealed a striking homology of both their nucleotide and deduced protein sequences, an identical genomic organization and high similarity in promoter architecture and mRNA expression pattern. Sequence analysis of the 5′‐flanking region of Pur‐1 revealed numerous potential binding sites for transcription factors Sp1, AP‐2 and Pur‐1/MAZ itself. An element required for basal Pur‐1 expression was mapped from nucleotide – 258 to + 43. This region also mediated stimulation of basal transcription by ectopically expressed MAZ protein. We conclude that the Pur‐1 gene is the murine homolog of human MAZ and, like it, belongs to the family of housekeeping genes.
The cyclic structure of microcin J25, a 21‐residue peptide antibiotic from Escherichia coli
Authors: Blond Alain , Péduzzi Jean , Goulard Christophe , Chiuchiolo María J. , Barthélémy Michel , Prigent Yann , Salomón Raúl A. , Farías Ricardo N. , Moreno Felipe , Rebuffat Sylvie
Published: March 2003

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non‐SOS‐dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol.174, 7428–7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin‐overproducing Escherichia coli strain by reverse‐phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two‐dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21‐residue peptide, cyclo(‐Val1‐Gly‐Ile‐Gly‐Thr‐Pro‐Ile‐Ser‐Phe‐Tyr‐Gly‐Gly‐Gly‐Ala‐Gly‐His‐Val‐Pro‐Glu‐Tyr‐Phe21‐). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21‐Val1 bond and led to a single peptide, MccJ25‐L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 µg·mL–1), the thermolysin‐linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N‐terminal extremity, the present study shows that removal of this extension and head–tail cyclization of the resulting propeptide are the only post‐translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.
Distinct regions specify the nuclear membrane targeting of emerin, the responsible protein for Emery–Dreifuss muscular dystrophy
Authors: Tsuchiya Yuichi , Hase Asako , Ogawa Megumu , Yorifuji Hiroshi , Arahata Kiichi
Published: March 2003

Emery–Dreifuss muscular dystrophy is a neuromuscular disorder that has three characteristics: (a) early contracture of the elbows, Achilles tendons and postcervical muscles; (b) slowly progressive wasting and weakness of skeletal muscle; and (c) cardiomyopathy with severe conduction block. The responsible gene for the X‐linked recessive form of this disease encodes an inner nuclear membrane protein named emerin. Although emerin is absent in tissues from patients with this disorder, it remains obscure why the loss of this widely expressed protein affects selectively skeletal muscle, heart and joints. As the first step to address this question, we examined the molecular regions of emerin that are essential for nuclear membrane targeting and stability of the protein. We found that the C‐terminal hydrophobic region was necessary, but not sufficient, for nuclear membrane anchoring and stability of the protein. In the absence of this transmembrane domain, the upstream nucleoplasmic domain showed no firm association with the nuclear rim, but showed the tendency to accumulate at the nucleolus‐like structures. Furthermore, proper targeting of emerin to the nuclear membrane required the latter half of the nucleoplasmic domain. These characteristics are distinct from those of lamina‐associated polypeptide 2. Our findings indicate that emerin has distinct interactions with the inner nuclear membrane components that may be required for the stability and function of rigorously moving nuclei in tissues such as skeletal muscle, heart and joints.
Molecular characterization of the B′ regulatory subunit gene family ofArabidopsisprotein phosphatase 2A
Authors: Haynes J. Georgia , Hartung Andrew J. , Hendershot John D. , Passingham R. Scott , Rundle Sabine J.
Published: March 2003

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants. We have identified a novelArabidopsisgene (AtB′δ) which encodes a 55‐kDa B′ type regulatory subunit of PP2A. The protein encoded by this gene is 57–63% identical and 69–74% similar to the previously identifiedAtB′genes. TheAtB′δgene appears to be expressed in allArabidopsisorgans indicating its protein product has a basic housekeeping function in plant cells. Unlike certain mRNAs derived from theAtB′γgene,AtB′δmRNAs do not fluctuate significantly in response to heat stress. Further analysis of cDNA sequences derived from theAtB′genes identified an alternatively spliced cDNA derived fromAtB′γ. This cDNA differs from the previously identifiedAtB′γcDNA by the absence of a 133‐bp region in its 5′ untranslated region. The missing 133‐bp region appears to constitute an unspliced intron and its presence in theAtB′γgene was confirmed by PCR usingArabidopsisgenomic DNA as a template.AtB′γmRNA containing the 133‐bp intron accumulate in allArabidopsisorgans and their levels fluctuate differentially in response to heat stress. The 133‐bp insert contains two short open reading frames and hence might serve as a translational control mechanism affecting AtB′γ protein synthesis. Finally we show, using both the yeast two hybrid system andin vitrobinding assays, that the B′ subunit ofArabidopsisPP2A is able to associate with other PP2A subunits, supporting the notion that the B′ protein serves as a regulator of PP2A activity in plants.
Structural heterogeneity of the binding sites of HSA for phenyl‐groups and medium‐chain fatty acids
Authors: Lund Martin , Bjerrum Ole J. , Bjerrum Morten J.
Published: March 2003

A new facet of the very heterogeneous albumin molecule is described. Chromatography at pH 6–9 of human serum albumin (HSA) on a phenyl‐sepharose column separates it into two nonconvertible conformations that are, in turn, in equilibrium with its binding and nonbinding forms. The hydrophobic interaction of HSA with phenyl‐sepharose depends on ionic strength, pH, and time of contact with the immobilized ligand. Binding as a function of pH shows a minimum at pH 6.5, and the binding profile at pH 7–9 fits the titration of a weak monoprotic acid with a pKa of 7.3. There was no observable difference in the CD spectra or the masses of the two forms. The equilibrium between the albumin forms was examined under defined conditions and cannot be explained by a simple two‐state model. Thus rechromatography of the nonbinding fraction derived from a sample in which 50% of the protein was originally retained resulted only in 10–20% bound protein. Correspondingly only 70–80% of the binding form was retained. A model explaining the observations can be derived if two species, I and II, exist in the solution, both being in an equilibrium with a binding and a nonbinding form, but in which I is not in equilibrium with II. The rate of conversion between the binding and nonbinding conformations was determined to be faster than 15 s at room temperature.
Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B‐276
Authors: Smith Thomas J. , Lloyd John S. , Gallagher Stephen C. , Fosdike William L. J. , Murrell J. Colin , Dalton Howard
Published: March 2003

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B‐276 is a three‐component enzyme system encoded by the four‐gene operon amoABCD. AMO catalyses the stereoselective epoxygenation of aliphatic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble methane monooxygenases of methanotrophic bacteria, to which AMO exhibits a significant degree of amino acid sequence identity. The AMO complex was not expressed in Escherichia coli, at least partly because that host did not produce all of the AMO polypeptides. Expression of AMO was achieved in Streptomyces lividans by cloning the AMO genes into the thiostrepton‐inducible expression plasmid pIJ6021. No background of AMO activity was detected in S. lividans cells without amoABCD and expression of AMO activity, at a level comparable to that from wild‐type R. rhodochrous B‐276, coincided with appearance of the AMO subunits. Recombinant AMO activity in cell‐free extracts of S. lividans was stimulated by the addition of NADH and produced R‐epoxypropane with comparable enantiomeric excess to AMO purified from the original organism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed individually in E. coli as fusions with glutathione S‐transferase. The expression systems described here now allow structure/function studies on AMO to be carried out by site‐directed mutagenesis.
Molecular cloning, expression and characterization of a monkey steroid UDP‐glucuronosyltransferase, UGT2B19, that conjugates testosterone
Authors: Bélanger Guy , Barbier Olivier , Hum Dean W. , Bélanger Alain
Published: March 2003

Although enzymatic processes involved in the formation of active steroids are well known, less information is available about the enzymes responsible for the metabolism of these hormones. Moreover, the expression of these catabolic enzymes, which include UDP‐glucuronosyltransferases, may play a role in the regulation of the level and action of steroid hormones in steroid target tissues. Previous studies have shown that the cynomolgus monkey contains high levels of circulating androgen glucuronides, indicating that it represents the best animal model to study the glucuronidation of steroids in extrahepatic tissues. Two cDNA libraries were constructed from monkey liver and prostate mRNA, and a novel UDP‐glucuronosyltransferase UGT2B cDNA, UGT2B19, was isolated from both libraries. The UGT2B19 cDNA is 2108 bp in length and contains an open reading frame of 1584 bp encoding a protein of 528 residues. The UGT2B19 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B19 protein was established. The activity of UGT2B19 on 3α‐hydroxy and 17β‐hydroxy positions of steroids was demonstrated. The enzyme also conjugates xenobiotics including eugenol, 1‐naphthol and p‐nitrophenol. Kinetic analysis revealed that UGT2B19 glucuronidates steroids with Km values of 1.6, 2.6 and 4.3 µm for testosterone, etiocholanolone and 5β‐androstane‐3α,17β‐diol, respectively. UGT2B19 transcript was detected, by specific reverse transcriptase‐PCR analysis in the liver, ovary, prostate, colon, spleen, kidney, pancreas, brain, cerebellum, mammary gland and epididymis. The molecular characterization of simian UGT2B19 demonstrates relevance of using monkey as an animal model to study and understand steroid glucuronidation in extrahepatic target tissue.
Enzymatic properties of vesicle‐reconstituted human cytochrome P450SCC (CYP11A1)
Authors: Kisselev Pyotr , Tuckey Robert C. , Woods Stephen T. , Triantopoulos Thrassi , Schwarz Dieter
Published: March 2003

The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys.353, 109–115] has enabled us to perform studies with the membrane‐reconstituted human enzymes to better understand the side‐chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane‐bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.