# Patent

## Title
Apoptosis-associated gene

## Abstract

A polypeptide possessing an action of causing chromatin condensation; a sense nucleic acid encoding the polypeptide; an antisense nucleic acid thereof, a probe or primer capable of specifically binding to the nucleic acid; an antibody or a fragment thereof against the polypeptide; an agent for controlling apoptosis, comprising the nucleic acid, the polypeptide, or the antibody or a fragment thereof; a screening method for a substance for controlling chromatin condensation, comprising evaluating an activity of causing chromatin condensation exhibited by the polypeptide; and a substance for controlling chromatin condensation. The present invention is useful for screening a substance controlling apoptosis, its use for controlling apoptosis and its applications to various diseases accompanying apoptosis.

## Background
This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/JP00/02254 which has an International filing date of Apr. 7, 2000, which designated the United States of America.
TECHNICAL FIELD
The present invention relates to an apoptosis-associated gene and a polypeptide, which are associated with the action mechanism of apoptosis, an agent for controlling apoptosis, a screening method for a substance for controlling chromatin-condensing activity, and the like.
BACKGROUND ART
In order that an organ or tissue functions normally in a living body, cell deaths of a part of cells as well as cell differentiation and cell proliferation are required. Most of such physiological cell deaths proceed due to apoptosis of which mechanism is usually strictly controlled.
Apoptosis is characterized by changes representatively including nuclear condensation and fragmentation of cells undergoing apoptosis, condensation and the fragmentation of the cells themselves, the fragmentation in the nucleosome unit (about 180 bp) of chromosomal DNA in the cells and the like.
For instance, in the formation process of an adult nematode (C. elegans), there is observed a phenomenon such that 131 cells die at a certain time in a certain site. In a mammal, it has also been known that a normal life event is maintained by the death of a certain cell at a certain time in the course of the development. These cell deaths are considered to be caused by apoptosis accompanied with morphological changes of the cells and the DNA fragmentation. Concretely, it has been shown that the cell deaths play an important role in the morphological formation during an individual development, the maintenance of a tissue homeostasis and the elimination of unwanted or hazardous cells.
Currently, studies on molecular mechanisms of apoptosis have been progressed. For instance, the studies on the action mechanisms of caspase-activated DNase [caspase-activated DNase (CAD)] and its inhibiting factor ICAD (inhibitor of CAD) in apoptosis have been made by the group of Nagata et al. [see, for instance, Enari, M., Nature, 391, 43–50 (1998) or the like]. Concretely, it is deduced that apoptosis signal causes an activation of cysteine protease caspase, and the resulting activated caspase then acts on CAD/ICAD complex (inactive form) to generate an active form CAD, and the active form CAD allows to fragmentate DNA into nucleosome unit, thereby resulting in cell death. Here, since the CAD synthesized in vitro in the absence of ICAD possesses no DNA-degrading activity, it is shown that the ICAD possesses a chaperonin function and is indispensable for the generation of the active form CAD.
Recently, it has been shown that the apoptosis is induced by a cancer-associated gene such as p53 antioncogene, c-myc or ras, an anti-cancer agent; irradiation with an ultraviolet or radioactive ray; or a certain cytokine representatively including Fas ligand, and the association with the signal transduction of apoptosis or the association with various diseases have come to a matter of interest.
However, studies on a target molecule for efficiently controlling apoptosis have not been sufficiently made at present.
DISCLOSURE OF INVENTION
An object of the present invention is to provide a polypeptide causing chromatin condensation without accompanying DNA fragmentation in nucleosome unit effective for controlling apoptosis, a sense or antisense nucleic acid encoding the polypeptide, an antibody against the polypeptide, and an agent for controlling apoptosis comprising the above-mentioned polypeptide, nucleic acid or antibody.
The gist of the present invention relates to:

    [1] a polypeptide possessing an action of causing chromatin condensation, having a sequence selected from the group consisting of:
    (A) the amino acid sequence of SEQ ID NO: 4; and
    (B) an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the sequence of SEQ ID NO: 4;
    [2] the polypeptide according to item [1] above, wherein the polypeptide possesses an action of causing chromatin condensation without accompanying DNA fragmentation;
    [3] a sense nucleic acid selected from the group consisting of:
    (a) a nucleic acid encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4;
    (b) a nucleic acid having the nucleotide sequence of SEQ ID NO: 8;
    (c) a nucleic acid encoding a polypeptide consisting of an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the sequence of SEQ ID NO: 4;
    (d) a nucleic acid having a nucleotide sequence having substitution, deletion, insertion or addition of at least one base in the nucleotide sequence of SEQ ID NO: 8; and
    (e) a nucleic acid capable of hybridizing to an antisense strand of a nucleic acid of any one of the above (a) to (d) under stringent conditions,

wherein the sense nucleic acid encodes a polypeptide possessing an action of causing chromatin condensation;

    [4] the sense nucleic acid according to item [3] above, wherein the polypeptide possesses an action of causing chromatin condensation without accompanying DNA fragmentation;
    [5] a polypeptide encoded by a nucleic acid selected from the group consisting of:
    (a) a nucleic acid encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4;
    (b) a nucleic acid having the nucleotide sequence of SEQ ID NO: 8;
    (c) a nucleic acid encoding a polypeptide consisting of an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the sequence of SEQ ID NO: 4;
    (d) a nucleic acid having a nucleotide sequence resulting from substitution, deletion, insertion or addition of at least one base in the nucleotide sequence of SEQ ID NO: 8; and
    (e) a nucleic acid capable of hybridizing to an antisense strand of a nucleic acid of any one of the above (a) to (d) under stringent conditions,

wherein the polypeptide possesses an action of causing chromatin condensation;

    [6] the polypeptide according to item [5] above, wherein the polypeptide possesses an action of causing chromatin condensation without accompanying DNA fragmentation;
    [7] an antisense nucleic acid corresponding to the sense nucleic acid of item [3] or [4] above;
    [8] a probe or primer capable of specifically binding to the sense nucleic acid of item [3] or [4] above, or to the antisense nucleic acid of item [7] above;
    [9] an antibody or a fragment thereof against the polypeptide of item [1] or [2] above, or the polypeptide of item [5] or [6] above;
    [10] an agent for controlling apoptosis, comprising the nucleic acid of any one of items [3], [4] and [7] above;
    [11] an agent for controlling apoptosis, comprising the polypeptide of item [1] or [2] above, or the polypeptide of item [5] or [6] above;
    [12] an agent for controlling apoptosis, comprising the antibody or a fragment thereof of item [9] above;
    [13] a screening method for a substance for controlling chromatin-condensing activity, comprising the step of evaluating an activity of causing chromatin condensation exhibited by the polypeptide of item [1] or [2] above, or the polypeptide of item [5] or [6] above, in the presence of a substance to be tested;
    [14] the screening method according to item [13] above, wherein an inhibition of an activity of causing chromatin condensation is evaluated;
    [15] the screening method according to item [13] above, wherein an induction of expression of an activity of causing chromatin condensation is evaluated;
    [16] the screening method according to item [13] above, wherein an enhancement of an activity of causing chromatin condensation is evaluated; and
    [17] a substance for controlling chromatin-condensing activity, which can be screened by the screening method of any one of items [13] to [16] above.





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph showing experimental results using an in vitro apoptosis system utilizing permeabilized HeLa cells. Permeabilized cells were incubated for 2 hours in the presence or absence of 0.1 mg/ml wheat germ agglutinin (WGA), an inhibitor of an active nuclear transport, by the use of an apoptotic Jurkat cell lysate (shown by “apoptosis” in the figure), a normal Jurkat cell lysate (shown by “normal” in the figure), a normal Jurkat cell lysate+caspase-3, or only with the casepase-3. After staining with Hoechst 33342, the nuclear morphologies were examined with a fluorescent microscope.
FIG. 2 is a photograph showing a profile of the separation of three distinct fractions each having an activity of inducing chromatin condensation in a bovine thymus lysate by a HiTrap Q column chromatography. Each fraction (2.5 μl) was assayed for its activity for inducing chromatin condensation using an in vitro system. Also, an upper panel of FIG. 2 shows chromatin condensation induced by each of three fractions (A, B and C). Further, a DNA ladder activity of each fraction was also assayed, and the results of an agarose gel electrophoresis are shown in a lower panel. A protein concentration was monitored by the absorbance at 280 nm.
FIG. 3 shows the results obtained by subjecting an aliquot of fraction having a chromatin-condensing activity at each purification step to SDS-PAGE, and silver-staining each product. Lane 1 shows a supernatant fraction (amount of protein: 3.4 μg) centrifuged at 100000×g; Lane 2 shows a HiTrap Q fraction (amount of protein: 1.7 μg); Lane 3 shows a fraction (amount of protein: 150 ng) obtained by passing through a hydroxyapatite column, and thereafter passing through a heparin sepharose; Lane 4 shows a Phenyl-Sepharose fraction (70 ng); Lane 5 shows a Superose 12 fraction (50 ng); and Lane 6 shows a MonoQ fraction (2.5 ng). An arrowhead indicates the electrophoretic position of purified Acinus p17 protein. A molecular weight marker (kDa) is given on the left.
FIG. 4 is a photograph showing the nuclear changes caused by the MonoQ fraction. HeLa cells were permeabilized and incubated with or without a MonoQ column chromatography-active fraction (right panel) or in the absence of caspase-3.
FIG. 5 shows a deduced amino acid sequence of human Acinus L (SEQ ID NO: 1). In this figure, the sequences of four kinds of peptides obtained from purified bovine Acinus p17 are underlined. An open triangle indicates an amino terminal of Acinus p17. Also, in the figure, a caspase-3 cleavage site is indicated by a solid triangle, and a P-loop site is indicated by a double-underline.
FIG. 6 is a schematic view showing Acinus. AcinusS has a unique sequence (MLSESKEG: hatched box)(SEQ ID NO: 9) at N-terminal subsequent to 767–1341 residues of AcinusL. AcinusS′ corresponds to 774–1341 residues of the AcinusL. ΔN and Acinus (987–1093) correspond to 987–1341 residues and 987–1093 residues, respectively, of the AcinusL. P-loop and the region homologous to RNA recognition motifs of S×1 are indicated by a solid box and a dotted box, respectively.
FIG. 7 shows the results of the comparison between Acinus and S×1 of Drosophila (SEQ ID NOS: 10 and 11). Identical amino acid residues and conserved amino acid residues are indicated by solid boxes and open boxes, respectively.
FIG. 8 shows the results of an in vivo cleavage of Acinus in Fas-mediated apoptosis. Jurkats cells were treated with 0.1 μg/ml anti-Fas antibody for an indicated time period (hours), and lyzed with a lysis buffer containing 1% Triton X-100. After centrifugation, the supernatant and a pellet were dissolved in SDS sample buffer to be subjected to SDS-PAGE. rAcinus resulting from removing His-tag by an enterokinase treatment of His-tagged Acinus (987–1093) expressed by using a vector (manufactured by NOVAGEN) employed for producing His-tagged protein was also subjected to SDS-PAGE at the same time. After SDS-PAGE, a protein band was transferred onto a membrane, and analyzed with an anti-Acinus antibody. The positions of the AcinusL, S, S′ and the cleaved fragments (85 kDa and 23 kDa) are indicated. The ratio (%) of the cells showing apoptotic chromatin condensation after nuclear treatment time is indicated in a lower column of the figure showing the electrophoretic results.
FIG. 9 shows the results of an in vitro cleavage of Acinus with caspase-3. Each of AcinusS and S (D/A) resulting from substitution of Asp1093 with Ala was transiently expressed in COS-7 cells. Next, AcinusS was incubated in the presence or absence of 10 μM DEVD-CHO and in the presence or absence of caspase-3 (2.5 ng) for 1 hour, subjected to SDS-PAGE and immunoblotted with an anti-Acinus antibody. The full length AcinusS and a caspase-cleaved product are indicated by a white arrowhead and a black arrowhead, respectively.
FIG. 10 is a graph showing the results of chromatin condensation induction by a recombinant Acinus in permeabilized cells. The induction by rAcinus of the chromatin condensation is dependent on caspase-3 and nuclear transport. Each of rAcinusΔN (about 0.05 μg), rAcinusΔN (D/A) (about 0.06 μg), rAcinus (987–1093) (0.1 μg) and rAcinusS (0.1 μg) was added to cells permeabilized in the presence or absence of caspase-3, DEVD-CHO and a nuclear transport protein as shown in the figure, and the mixture was incubated for 2 hours. rAcinusΔN was preincubated with caspase-3 at 37° C. for 1 hour. After staining with Hoechst 33324, the chromatin condensation was evaluated with a fluorescent microscope. The data are shown as means±s.d (n=4).
FIG. 11 is a photograph showing the nuclear localization of Acinus in permeabilized cells, and in intact cells. Permeabilized HeLa cells were incubated with each of His-tagged rAcinusΔN and ΔN (D/A) and then immunostained with each of an anti-Xpress antibody and an FITC-labelled secondary antibody. The intact HeLa cells were immunostained with an anti-Acinus antibody and then an RITC-labelled secondary antibody. Further, the nucleus was visualized by means of Hoechst 33342 staining. Each photomicrograph is obtained from an identical field of vision.
FIG. 12 shows the results obtained by incubating permeabilized Jurkat cells, in the presence or absence of caspase-3 together with a normal Jurkat cell lysate or rAcinusΔN for 2 hours, and subjecting the nuclear DNA to electrophoresis on agarose gel.
FIG. 13 shows the results of immunodepletion of Acinus derived from a Jurkat cell lysate. Each of the untreated Jurkat cell lysate (indicated by “normal lysate” in the figure) and the Jurkat cell lysate treated with an anti-Fas antibody for three hours (indicated by “apoptosis lysate” in the figure) were incubated with a control IgG antibody- or an anti-Acinus antibody-conjugated protein G sepharose. A treated lysate was separated by SDS-PAGE and immunoblotted with an anti-Acinus antibody. Further, a whole cell extract from normal Jurkat cells dissolved in an SDS sample buffer was loaded onto the leftmost lane for the purpose of identifying the band of endogenous Acinus. Each arrowhead indicates the band of the AcinusL, S, S′ and Acinus (987–1093). An arrow indicates one of the unidentified proteins different from Acinus (987–1093).
FIG. 14 is a photograph showing that Acinus-immunodepletion lysate does not induce chromatin condensation and recovers its activity by rAcinusΔN. An immunodepleted lysate (5 μl) derived from live cells (indicated by “normal lysate” in the figure) immunodepleted by an indicated antibody was added together with or without the rAcinusΔN (0.5 μg) to permeabilized cells in the presence of caspase-3. An immunodepleted lysate derived from an anti-Fas antibody-treated cell (indicated by “apoptosis lysate” in the figure) was added in the absence of caspase-3. The nucleus was visualized by means of Hoechst 33342 staining. The ratio (%) of the nucleus exhibiting chromatin condensation is indicated under each fluorescent photomicrograph.
FIG. 15 shows the enhancement and the retardation of chromatin condensation in HeLa cells transfected with acinusL plasmid in the sense direction and the antisense direction. 1 μg of pCAGGS-acinusL (sense: indicated by solid circles in the figure), pCAGGS-acinusL (R) (antisense: indicated by open circles in the figure) or a vector (indicated by x in the figure) was transfected into HeLa cells together with a GFP-expressing plasmid (0.1 μg). After 48 hours, the cells were further incubated for an indicated time period in the presence of 100 μM etoposide. The chromatin condensation in GFP-positive cells was evaluated by means of Hoechst 33342 staining. The results are shown as means±s.d (n=4).
FIG. 16 shows the reduction in endogenous Acinus by the transfection with acinusL (R) antisense plasmid. HeLa cells (0.5×106) were transfected with 1 μg of a pCAGGS-acinusL (R) plasmid or only with a vector. After 48 hours, the cells were harvested, and the whole cell extract was prepared by use of an SDS sample buffer. A sample was subjected to SDS-PAGE, and then immunoblotted with an anti-Acinus antibody (left panel) and an anti-Ran antibody (right panel). The AcinusL, S, S′ and Ran were indicated by arrowheads.



BEST MODE FOR CARRYING OUT THE INVENTION
In the present specification, the term “apoptosis” used herein means a way how cells die, characterized by changes representatively including condensation and fragmentation of nuclear chromatin of the cells, the condensation and the fragmentation of such cells themselves and the fragmentation of the chromosomal DNA in the nucleosome unit (about 180 bp) of the cells, including an event caused by a pathological factor such as a disease as well as a physiological factor (for example, expression of a physiological event such as immune, hormone action and development).
The above-mentioned morphological changes by apoptosis comprise the following stages. First, cells shrink and separate from adjacent cells, and chromatin, which is a complex of nuclear DNA with a protein, undergoes condensation in the periphery of the nuclear membrane, resulting in the nuclear condensation. At the same time, cilia on the cell surface disappear imparting a smoothened surface, and irregular projections appear and then cells are twisted and torn, to be fragmented into membrane-inclusion spherical apoptotic small bodies having various sizes. Subsequently, the resulting apoptotic small bodies are subjected to phagocytosis by phagocytic cells such as macrophage. The above-mentioned morphological changes are expressed following an identical morphological process regardless of the kinds of the cells, the kinds of the organisms or the inducing factors.
One of the significant features of the polypeptide of the present invention resides in that the polypeptide comprises a polypeptide sequence possessing an action for causing chromatin condensation. The chromatin condensation is caused without accompanying DNA fragmentation in nucleosome unit.
The evaluation of the chromatin condensation can be carried out by adding a standard assay mixture (5 μl) [composition: ATP generating system (composition: 1 mM ATP, 5 mM creatine phosphate and 20 units/ml creatine phosphate kinase), 1 mM GTP, 50 ng/ml recombinant caspase-3, 0.5 mg/ml importin α, 0.5 mg/ml importin β, 0.1 mg/ml Ran, 10 ng/ml p10, and the polypeptide (0.05 μg) of the present invention] to permeabilized HeLa cells or to 1 μl of permeabilized Jurkat cells (1×106) on a plate, and then incubating the resulting mixture at 37° C. for 2 hours, and evaluating the nuclear changes under a fluorescent microscope after staining with 10 μM Hoechst 33342.
Also, the detection of DNA fragmentation can also be carried out as described by Enari et al. [Enari, M., EMBO J. 14, 5201–5208 (1995)].
In the preparation of the permeabilized HeLa cells which are used in the above-mentioned evaluation of the chromatin condensation, HeLa cells grown on a plate are subjected to a modified Adam's method [Adam et al., J. Cell. Biol. 111, 807–816 (1990)], whereby the permeability of HeLa cells can be improved. Concretely, the cells are washed with a transport buffer, treated with 20 μg/ml of digitonin (manufactured by Wako Pure Chemical Industries, Ltd.) for 3 minutes at room temperature, and then immersed in the transport buffer for 5 minutes, whereby the permeability can be improved. Also, in Jurkat cells, the permeability can be improved in accordance with the method of Gorlich et al. [Gorlich et al., Cell 79, 767–778 (1994)].
In the present specification, the phrase “polypeptide possessing an action for causing chromatin condensation” means a polypeptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, the polypeptide chain being capable of exhibiting an ability of causing chromatin condensation under conditions without proteolytic effect, for example, under the conditions in the absence of caspase-3 and in the presence of an inhibitor for inhibiting caspase. The polypeptide causing chromatin condensation described above can cause chromatin condensation without accompanying DNA fragmentation.
The polypeptide having the amino acid sequence of SEQ ID NO: 1 is a human Autholog of a factor which has been found for the first time in a HiTrap Q fraction of a bovine thymus cell lysate, and it has never been conventionally expected that a polypeptide sequence inducing apoptotic chromatin condensation exists in a part of the sequence. The associated factor of the polypeptide causing chromatin condensation is named Acinus (Apoptotic chromatin condensation inducer in the nucleus). Especially, a factor consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 1 is designated as AcinusL.
It is suggested that in the downstream of an effector caspase-3, there are at least three different pathways as pathways involved in apoptotic changes in the nucleus, including (1) caspase-6 known as a lamin protease which breaks a nuclear membrane structure, (2) CAD/DEF40 causing an oligonucleosomal DNA cleavage and (3) Acinus inducing chromatin condensation without DNA fragmentation in oligonucleotide units, and thus provides a novel target in the control of apoptosis.
The above-mentioned phrase “polypeptide possessing an action of causing chromatin condensation” includes, for instance, Acinus derivative as indicated by (987–1093) in FIG. 6 [referred to as Acinus (987–1093)] and the like. Acinus (987–1093) mentioned above is a polypeptide having the amino acid sequence (SEQ ID NO: 4) of positions 987–1093 in the above-mentioned amino acid sequence of SEQ ID NO: 1, which can be said to be an active form polypeptide of Acinus, because the polypeptide causes chromatin condensation even in the absence of caspase-3 and in the presence of a caspase inhibitor. More concrete examples of the above-mentioned “polypeptide possessing an action of causing chromatin condensation” include a polypeptide possessing an action of causing chromatin condensation, having a sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 4, and an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 4. Among them, a polypeptide possessing an action of causing chromatin condensation without accompanying DNA fragmentation is preferred. The polypeptide can be selected by evaluating the characteristics in accordance with “evaluation for chromatin condensation” described above, and “detection for DNA fragmentation” described above as occasion demands.
In the present invention, the phrase “amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue” may be a naturally occurring sequence, or it may be an artificially prepared sequence, for instance, a sequence prepared from a nucleic acid described below by means of genetic engineering techniques.
The number of substitution, deletion, insertion or addition of the above-mentioned amino acids may be such that the resulting polypeptide is a polypeptide possessing an action of causing chromatin condensation, more preferably a polypeptide possessing an action of causing chromatin condensation without accompanying DNA fragmentation.
Besides the above-mentioned polypeptide having the sequence of SEQ ID NO: 4, there may be considered a case where the polypeptide possessing an action of causing chromatin condensation exists among the polypeptides which can be produced by a mechanism existing in a living body, such as an RNA differential splicing or a proteolytic action from genomic DNA corresponding to a cDNA encoding the polypeptide consisting of the amino acid of SEQ ID NO: 1, and such a polypeptide is also encompassed by the present invention. The polypeptide can be selected by evaluating the characteristics in accordance with “evaluation for chromatin condensation” described above, and “detection for DNA fragmentation” described above as occasion demands.
Concretely, each of AcinusL mentioned above, AcinusS of SEQ ID NO: 2 and AcinusS′ of SEQ ID NO: 3 is considered to be a precursor which can be converted in a living body into an active form by a differential splicing and/or an action of caspase-3.
Further, a polypeptide encoded by a nucleic acid capable of hybridizing to an antisense strand of any of the nucleic acids (a) to (d) described below under stringent conditions, the polypeptide possessing an action of causing chromatin condensation is also encompassed by the polypeptide of the present invention. The polypeptide can be selected by evaluating the characteristics in accordance with “evaluation for chromatin condensation” described above, and in combination with “detection for DNA fragmentation” described above as occasion demands.
The polypeptide of the present invention can be obtained by allowing to express a nucleic acid described below by a known method, and then performing a known separation method. Concrete examples thereof are given in Examples, which are not limitative to the Examples in any way.
The polypeptide of the present invention can be used, for instance, for screening for a substance capable of controlling the action of the polypeptide, screening for a substance controlling apoptosis and control of apoptosis described below.
One of the significant features of the nucleic acid of the present invention resides in that the nucleic acid encodes the above-mentioned polypeptide possessing an action of causing chromatin condensation. The above-mentioned polypeptide encoded by the nucleic acid can cause chromatin condensation without accompanying DNA fragmentation.
In the present specification, the term “nucleic acid” refers to genomic DNA, cDNA, RNA and a nucleic acid analog. Here, the term “nucleic acid analog” refers to bases constituting the nucleic acid, such as cytosine, guanine, thymine, adenine and uracil and/or those with modifications in the sugar backbone.
The “nucleic acid encoding the polypeptide causing chromatin condensation” includes cDNA of a sense strand encoding the polypeptide of the present invention causing chromatin condensation without accompanying the DNA fragmentation, a corresponding RNA, a nucleic acid analog sequence, and the like.
Concretely, the nucleic acid includes sense nucleic acids selected from the group consisting of:

    (a) a nucleic acid encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (sequence corresponding to positions 987–1093 in the amino acid sequence of SEQ ID NO: 1);
    (b) a nucleic acid having the nucleotide sequence of SEQ ID NO: 8;
    (c) a nucleic acid encoding a polypeptide consisting of an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the sequence of SEQ ID NO: 4;
    (d) a nucleic acid having a nucleotide sequence having substitution, deletion, insertion or addition of at least one base in the nucleotide sequence of SEQ ID NO: 8; and
    (e) a nucleic acid capable of hybridizing to an antisense strand of a nucleic acid of any one of the above (a) to (d), under stringent conditions,

wherein the sense nucleic acid encodes a polypeptide causing chromatin condensation.



The nucleic acid encoding a polypeptide consisting of an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the amino acid sequence, and the DNA having a nucleotide sequence having substitution, deletion, insertion or addition of at least one base in the nucleotide sequence can be prepared by the method described in Molecular Cloning: A Laboratory Manual, 2nd Ed. [Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)] or the like, which can be prepared by, for instance, the method of site-directed mutagenesis, the PCR method, and the like. In the present invention, the number of substitution, deletion, insertion or addition of the amino acid may be such that the polypeptide to be expressed is within the scope of a polypeptide capable of generating a polypeptide causing chromatin condensation.
Further, the present invention also encompasses a nucleic acid capable of capable of hybridizing to an antisense strand of a nucleic acid mentioned above, under stringent conditions, so long as the polypeptide to be expressed possesses an action of causing chromatin condensation.
Here, the “stringent conditions” include hybridization conditions described in Molecular Cloning: A Laboratory Manual, 2nd Ed. or the like. Concretely, the hybridization conditions include conditions of carrying out heating at 42° C. in a solution containing 6×SSC, 0.5% SDS and a 50% formamide solution, and thereafter washing at 68° C. in a solution containing 0.1×SSC and 0.5% SDS.
Further, the present invention also encompasses a nucleic acid capable of generating the nucleic acid of any one of (a) to (e) mentioned above.
Here, the phrase “nucleic acid capable of generating a nucleic acid of any one of (a) to (e)” means a nucleic acid comprising a nucleic acid of any one of (a) to (e), and capable of generating the nucleic acid of any one of (a) to (e) by transcription of RNA to genomic DNA, splicing or the like.
Concrete examples thereof include sense nucleic acids selected from the group consisting of:

    (f) a nucleic acid encoding a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 3;
    (g) a nucleic acid having the nucleotide sequence of any one of SEQ ID NOs: 5 to 7;
    (h) a nucleic acid encoding a polypeptide consisting of an amino acid sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the amino acid sequence of any one of SEQ ID NOs: 1 to 3;
    (i) a nucleic acid having a nucleotide sequence having substitution, deletion, insertion or addition of at least one amino acid residue in the nucleotide sequence of any one of SEQ ID NOs: 5 to 7; and
    (e) a nucleic acid capable of hybridizing to an antisense strand of the nucleic acid of any one of the above (a) to (d) under stringent conditions,

wherein the sense nucleic acid encodes a polypeptide capable of generating a polypeptide causing chromatin condensation without accompanying chromatin condensation. Incidentally, each of SEQ ID NOs: 5 to 7 mentioned above corresponds to human acinusL, acinusS and acinusS′.



Also, the present invention encompasses a nucleic acid capable of hybridizing to an antisense strand of the nucleic acid under stringent conditions, so long as the polypeptide to be expressed possesses an action of causing chromatin condensation.
The present invention also encompasses a polypeptide encoded by the nucleic acid of the present invention, so long as the resulting polypeptide is a polypeptide possessing an action of causing chromatin condensation.
Also, the present invention also encompasses an antisense nucleic acid corresponding to the above-mentioned sense nucleic acid.
The nucleic acid can be used for control of apoptosis described below, screening of an autholog originated from another organism or the like.
The polypeptide of the present invention can be mass-produced by expressing the nucleic acid (sense nucleic acid) of the present invention.
A protein resulting from expression of a nucleic acid can be produced on the bases of many textbooks and literatures, for instance, Molecular Cloning: A Laboratory Manual, 2nd Ed. mentioned above or the like. An expression plasmid capable of replicating thereof and functioning in host cell by adding a translation initiation codon in the upstream of the nucleic acid to be expressed and a translation stop codon to the downstream thereof, adding a regulatory gene such as a promoter sequence for regulating transcription (for instance, trp promoter, lac promoter, T7 promoter, SV40 early promoter), and incorporating the resulting gene in an appropriate vector (for instance, pBR322, pUC19, pSV SPORT1 or the like).
Next, the transformant cells are obtained by introducing an expression plasmid into appropriate host cells. The host cells include cells of a prokaryote such as Escherichia coli, a unicellular eucaryote such as an yeast, a multicellular eucaryote such as an insect and an animal, and the like. The method for incorporating a gene into a host cell includes calcium phosphate method, DEAE-dextran method, electric pulse method, and the like. The transformant produces a desired protein by culturing in an appropriate medium. The protein obtained as described above can be isolated and purified by a general biochemical method. In addition, in order to facilitate isolation and purification, there may be added a sequence which can be expressed as His tag, or GST fusion protein.
There is provided a probe or primer capable of specifically binding to the above-mentioned sense nucleic acid or antisense nucleic acid by the use of the nucleic acid of the present invention.
Here, the phrase “probe or primer capable of specifically binding to (the nucleic acid)” encompasses an oligonucleotide capable of hybridizing under the hybridization conditions which are suitable for the oligonucleotide to be used as the probe or primer.
The length of the above-mentioned probe or primer and the nucleotide sequence can be appropriately selected from the sequences of the nucleic acid of the present invention in consideration of the Tm values depending upon its purpose of use. It is desired that the length of the above-mentioned probe is 14 nucleotide length or more, preferably 18 nucleotide length or more, from the viewpoint of preventing nonspecific hybridization. In addition, it is desired that the length of the primer is, for instance, 15 to 40 nucleotide length, preferably 17 to 30 nucleotide length.
The above-mentioned probe or primer can be usually prepared by a method used in nucleic acid synthesis. For instance, the probe or primer can be prepared by chemical synthesis method representatively including phosphoramidite method, and an enzymatic synthesis method utilizing DNA polymerases.
In addition, the probe or primer can be prepared by fragmenting the nucleic acid of the present invention by an enzymatic treatment with a restricted endonuclease, and various nucleases, or a short physical treatment representatively including sonication treatment, and isolating the resulting fragment.
The conditions for the specific binding in the above-mentioned probe or primer include the hybridization conditions described in Molecular Cloning: A Laboratory Manual, 2nd Ed. mentioned above or the like. Such conditions include, for instance, conditions of 0.1×SSC and 65° C. in a case of a sufficiently long nucleotide; and conditions of 6×SSC and 25° C. in a case of a short nucleotide; and the like.
The above-mentioned probe or primer is thought to be applied to screening of various libraries (genomic DNA or cDNA), pharmaceuticals, research reagents, and the like.
The antibody of the present invention is not particularly limited, so long as the antibody possesses an ability of specifically binding to Acinus, which is the concrete polypeptide of the present invention. The antibody may be any of polyclonal antibodies and monoclonal antibodies, and it may be a fragment thereof. Further, antibodies modified by known techniques and antibody derivatives, for instance, humanized antibody, Fab fragment, single-chain antibody, and the like can be also used. The antibody of the present invention can be readily prepared by appropriately immunizing a rabbit, a mouse or the like using all or a part of the polypeptide of the present invention in accordance with the method described in, for instance, Current Protocols in Immunology, edited by John E. Coligan, published by John Wiely & Sons, Inc., 1992. In addition, the antibody can be prepared by genetic engineering means. In addition, the antibody encompasses an antibody capable of specifically binding to a partial fragment of the polypeptide, or a fragment thereof.
The resulting antibody is purified and thereafter treated with a peptidase or the like, thereby giving an antibody fragment. The use of the resulting antibody or a fragment thereof includes applications to affinity chromatography, screening of various kinds of libraries (genomic DNA or cDNA), pharmaceuticals, research reagents, and the like.
Further, when the antibody of the present invention is used in enzyme immunoassay, fluorescent immunoassay or luminescent immunoassay, the antibody may also be modified in various ways for the purpose of facilitating the detection.
According to the present invention, there may further be provided an agent for controlling apoptosis.
One of the significant features of the agent for controlling apoptosis of the present invention resides in that the agent for controlling apoptosis comprises the polypeptide, the nucleic acid or the antibody of the present invention. As described above, since the agent for controlling apoptosis comprises the polypeptide, the nucleic acid or the antibody, there is exhibited an excellent effect such that chromatin condensation in apoptosis can be suppressed or accelerated. The agent for controlling apoptosis of the present invention will be described below in individual embodiments of (1) an agent for controlling apoptosis comprising a polypeptide, (2) an agent for controlling apoptosis comprising a nucleic acid, (3) an agent for controlling apoptosis comprising an antibody, and (4) an embodiment other than the agents for controlling apoptosis of embodiments (1) to (3) described above.
(1) Agent for Controlling Apoptosis Comprising Polypeptide
In the agent for controlling apoptosis of the present invention, the polypeptide possessing an action of causing chromatin condensation of the present invention can be employed as it is, or in a form subjected to various modifications for facilitating the incorporation into a certain cell.
While an in vivo immune response may sometimes be caused when an agent for controlling apoptosis comprising the polypeptide is employed, various aids for reducing the immune response may be added within the range such that the polypeptide of the present invention exhibits an action for causing chromatin condensation.
(2) Agent for Controlling Apoptosis Comprising Nucleic Acid
In the agent for controlling apoptosis of the present invention, a sense nucleic acid and an antisense nucleic acid can appropriately be selected depending upon the purpose of use.
For example, when the apoptosis takes place by causing the chromatin condensation, a sense nucleic acid is used. On the other hand, when apoptosis is suppressed by suppressing chromatin condensation, an antisense nucleic acid is used.
The method of administering the agent for controlling apoptosis comprising the sense nucleic acid of the present invention to be introduced into cells includes a method of administering a construct resulting from incorporation of the nucleic acid into a viral vector as the agent for controlling apoptosis.
The viral vector includes, for instance, RNA viruses and DNA viruses such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, and the like.
Other methods include a method of directly intramuscularly administering an agent for controlling apoptosis comprising an expression plasmid harboring a sense nucleic acid of the present invention (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a potassium phosphate method, an electroporation method and the like.
The agent for controlling apoptosis can be administered via a suitable administration route depending on the target of which apoptosis is to be controlled. For example, the agent for controlling apoptosis can be intravenously, arterially, subcutaneously or intramuscularly administered. When the agent is administered by in vivo method, the agent can, for instance, take a form of preparations such as liquid preparations. In general, the agent takes the form of an injection or the like comprising the nucleic acid of the present invention as an active ingredient, and a conventional vehicle may be added thereto as occasion demands. In a liposome or membrane fusion liposome comprising the nucleic acid of the present invention (such as Sendai virus (HVJ)-liposome), the agent can take a form of liposome preparations such as suspensions, cryogens and centrifugally-concentrated cryogens.
The content of the nucleic acid of the present invention in the preparation can be properly adjusted depending on the target of which apoptosis is to be controlled.
When the antisense nucleic acid is employed, the antisense nucleic acid can, for instance, be prepared on the basis of the nucleotide sequence of the sense nucleic acid encoding the polypeptide of the present invention, or a corresponding antisense nucleic acid can be readily prepared by incorporating the sense nucleic acid into a gene expression plasmid in the antisense direction.
This antisense oligonucleotide may be a sequence complementary to any of the parts of the coding part or 5′ non-coding part of a cDNA, which is the nucleic acid of the present invention. It is desired that the antisense oligonucleotide is a sequence complementary preferably to transcription initiation site, translation initiation site, 5′ non-translation region or exon regions.
The term chemically-modified product of a nucleic acid refers to a chemically-modified product capable of enhancing transition ability or stability of DNA or RNA in a cell. The chemically-modified product includes, for instance, derivatives such as phosphothioate, phosphorodithioate, alkyl phosphotriesters, alkyl phosphonates, alkyl phosphoamidates and the like [Antisense RNA and DNA, published by WILEY-LESS, 1–50 (1992)]. This chemically-modified product can be prepared in accordance with the literature mentioned above.
When an expression plasmid into which an antisense nucleic is incorporated is used as an agent for controlling apoptosis, the agent for controlling apoptosis may be administered to a target by a method utilizing a liposome, a recombinant virus and the like. The expression plasmid of an antisense nucleic acid can be simply prepared by ligating the sense nucleic acid of the present invention in such a manner that a transcription is carried out in an opposite direction downstream of a promoter by using an usual expression vector.
To the agent for controlling apoptosis comprising an antisense nucleic acid or a chemically-modified form thereof as it is, various aids can be added, within the range such that the agent exhibits an action of causing chromatin condensation.
(3) Agent for Controlling Apoptosis Comprising Antibody
In the agent for controlling apoptosis comprising an antibody, various adjuvants may be contained so that Acinus in the cells can be depleted. Its preparation form is not particularly limited and can be appropriately determined depending on the target of which apoptosis is to be controlled, the purpose of use, and the like. The content of the antibody of the present invention in the agent for controlling apoptosis can be appropriately determined depending on the target of which apoptosis is to be controlled.
Furthermore, a nucleic acid capable of expressing the antibody of the present invention in the cells can be prepared, and the resulting nucleic acid can be used similarly to the administration of the agent for controlling apoptosis comprising the nucleic acid mentioned above.
(4) Embodiment Other than Agents for Controlling Apoptosis of Embodiments (1) to (3)
Also, a substance for controlling the action of the polypeptide of the present invention can also be screened by using the polypeptide of the present invention or the like, and the resulting substance can also be used as the agent for controlling apoptosis of the present invention.
Further, a substance for controlling transcription and translation from the nucleic acid of the present invention, for instance, ribozyme, and a nucleic acid for forming a triple stranded-nucleic acid with a double-stranded DNA encoding a region encoding expression can also be used as the agent for controlling apoptosis of the present invention.
The term “ribozyme” refers to an RNA molecule possessing an activity of cleaving mRNA which encodes a certain protein, and inhibiting expression of the particular protein. The ribozyme can be designed on the basis of the nucleotide sequence encoding a certain protein. For instance, as a hammerhead ribozyme, one obtained by a method described in FEBS Letter, 228, 228–230 (1988) can be used. In addition to the hammerhead ribozyme, the ribozyme as referred to in the present specification encompasses any of those ribozymes, regardless of the kinds of the ribozymes, such as hairpin-shaped ribozymes and delta-shaped ribozymes, so long as the ribozyme is capable of cleaving mRNA of a particular protein, thereby inhibiting expression of the particular protein.
The nucleic acid for forming a triple-stranded nucleic acid with a double-stranded DNA encoding a region suppressing expression can be prepared by referring to, for instance, Nucleic Acids Research, 19, 3435–3441.
The agent for controlling apoptosis of the present invention is expected to be further applicable to various diseases accompanied with apoptosis.
The diseases accompanied with apoptosis include AIDS, neurodegenerative diseases (for instance, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa cerebellar degeneration and the like), osteomyelodysplastic diseases (for instance, aplastic anemia and the like), ischemic diseases (for instance, myocardial infarction, apoplexy and the like), hepatic diseases caused by intoxication with an alcohol or the like, cancers, autoimmune diseases (for instance, systemic lupus erythematosus, immune-associated glomerulonephritis and the like), viral infections (for instance, herpes viral infection, adenoviral infection and the like), diabetes, viral hepatitis and the like.
For instance, when the sense nucleic acid is used, apoptosis can be controlled positively (increase of apoptosis), so that effects for cancers, autoimmune diseases (for instance, systemic lupus erythematosus, immune-associated glomerulonephritis and the like) and viral infections (for instance, herpes viral infection, adenoviral infection and the like) are expected.
When the antisense nucleic acid or antibody is used, apoptosis can be controlled negatively (decrease of apoptosis), so that effects for AIDS, neurodegenerative diseases, osteomyelodysplastic diseases, ischemic diseases and hepatic diseases caused by intoxication with an alcohol or the like are expected.
One of the significant features of the method for controlling apoptosis of the present invention resides in the use of the above-mentioned agent for controlling apoptosis against mammals (for instance, cattle and the like). According to the method of the present invention, apoptosis can positively or negatively be controlled by selecting a peptide, a nucleic acid and an antibody to be contained in the above-mentioned agent for controlling apoptosis.
The method for controlling apoptosis of the present invention is expected to be further applicable to various diseases accompanied with apoptosis.
The present invention further provides a screening method for a substance for controlling chromatin-condensing activity.
The screening method of the present invention is concretely a screening method for a substance for controlling chromatin-condensing activity, comprising the step of evaluating an activity of causing chromatin condensation of the polypeptide of the present invention in the presence of a substance to be tested.
The activity of causing chromatin condensation can be determined in accordance with the evaluation of chromatin condensation described above.
The substance for inhibiting chromatin-condensing activity can be screened by evaluating the inhibition of an activity of causing chromatin condensation.
An expression inducer for chromatin-condensing activity can be screened by evaluating the induction of expression of an activity of causing chromatin condensation.
The enhancer for chromatin-condensing activity can be screened by evaluating the activity of causing chromatin condensation in the presence or absence of a substance to be tested.
According to the screening method of the present invention, a substance for controlling chromatin-condensing activity, such as a substance for inhibiting chromatin-condensing activity, an inducer for expressing chromatin-condensing activity or an enhancer for chromatin-condensing activity, can be screened. The substance for controlling chromatin-condensing activity is also encompassed by the present invention.
The present invention will be explained in further detail by means of the following examples, without intending to limit the present invention to the scope of the examples.
The reagents, the cells, and the like used in the following examples are given below.
Reagents Used and Cells Used
HeLa (D28/AH2) cells and Jurkat cells were grown in RPMI 1640 containing 10% fetal bovine serum. Bovine thymus glands were obtained from a slaughterhouse. Recombinant active human caspase-3 was produced and purified using the Xpress system (manufactured by Invitrogen), and was dialyzed against a transport buffer (composition: 20 mM HEPES, pH 7.3, 110 mM CH3COONa, 0.5 mM EGTA, and 2 mM dithiothreitol). Recombinant human p10, human Ran, human importin α and importin β were purified as described in [Imamoto, N. et al., EMBO J. 14, 3617–3626 (1995); Tachbana, T. et al., FEBS Lett. 397, 177–182 (1996); Melchior, F. et al., Meth. Enzymol. 257, 279–291 (1995)]. An anti-human Fas monoclonal antibody (CH-11) and an anti-Xpress antibody were obtained from MBL and Invitrogen, respectively. A polyclonal rabbit antibody against human Acinus was generated against a synthetic peptide corresponding to amino acid residues 987–1000 of AcinusL. DEVD-CHO was obtained from Osaka Peptide Institute. Hydroxyapatite was obtained from SEIKAGAKU CORPORATION, and all other column carriers used for protein purification were obtained from Pharmacia.
EXAMPLE 1
1) Preparation of Cell Lysate for In Vitro Apoptosis Assay
Jurkat cells (4×108) treated with anti-Fas antibody CH-11 (0.1 μg/ml) for 3 hours and untreated cells were sonicated in 0.5 ml of lysis buffer (composition: 5 mM HEPES, pH 7.3, 10 mM CH3COOK, 2 mM (CH3COO)2Mg, 5 mM CH3COONa, 0.5 mM EGTA, 2 mM dithiothreitol, 10 μg/ml cytochalasin B, 50 μg/ml APMSF, and 1 μg/ml each of aprotin, leupeptin and pepstatin) and centrifuged. The resulting supernatant was concentrated with an ultrafiltration membrane-MC5,000 NMWL filter unit (manufactured by Millipore Corporation) to 20–30 mg protein/ml.
2) In vitro Apoptosis Assay with Permeabilized Cells
HeLa cells grown on plates were permeabilized as described by Adam et al. [Adam et al., J. Cell Biol. 111, 807–816 (1990)] with some modifications. Concretely, cells were rinsed with the transport buffer, treated with 20 μg/ml digitonin (manufactured by Wako Pure Chemicals Industries, Ltd.) for 3 minutes at room temperature, and then immersed in the transport buffer for 5 minutes. In Jurkat cells, the permeability can be improved in accordance with the method of Gorlich et al. [Gorlich et al., Cell 79, 767–778 (1994)]. The standard assay mixture (5 μl) was constituted by an ATP-regeneration system (composition: 1 mM ATP, 5 mM creatine phosphate, and 20 units/ml creatine phosphokinase), 1 mM GTP, 50 ng/ml of recombinant caspase-3, 0.5 mg/ml importin α, 0.5 mg/ml importin β0.1 mg/ml Ran, 10 ng/ml p10, and the indicated column fractions. In the case of whole lysate (4.5 μl), only the ATP-regeneration system was added. The reaction was started by addition of the mixture to permeabilized HeLa cells on plates or to 1 μl of permeabilized Jurkat cells (1×106 cells), followed by incubation at 37° C. for 2 hours. After staining with 10 μM Hoechst 33342, the nuclear changes were evaluated under a fluorescent microscope. The detection of DNA fragmentation in permeabilized Jurkat cells was carried out as described in accordance with the method by Enari et al. [Enari, M., EMBO J. 14, 5201–5208 (1995)].
3) Immunofluorescence Microscopy
HeLa cells grown on coverslips and permeabilized cells were fixed for 10 minutes with 3.7% formaldehyde in PBS. After treatment with 0.1% Triton X-100 in PBS for 5 minutes, the cells were incubated overnight together with 1 μg of an anti-Acinus antibody or anti-Xpress antibody in PBS containing 5% skim milk, and then were incubated with the RITC— or FITC-labeled secondary antibody.
4) Molecular Search Acting on Downstream of Caspase Capable of Responding to Nuclear Changes by Apoptosis
As shown in FIG. 1, when the permeabilized HeLa cells and the lysate prepared from apoptotic Jurkat cells were incubated, apoptotic morphological changes of the nucleus, including apoptotic chromatin condensation and the like were induced in nearly all nuclei. As shown in FIG. 1, the lysate derived from live Jurkat cells treated with caspase-3 also induced similar phenomenon to the chromatin condensation in the lysate, whereas untreated lysate or caspase-3 alone did not induce any nuclear changes, thereby suggesting that the target molecule of caspase-3 responsible for chromatin condensation was present in the lysate. Further, as shown in FIG. 1, chromatin condensation was inhibited by the addition of wheat germ agglutinin, thereby suggesting the involvement of active nuclear transport.
EXAMPLE 2
Purification of Factor (Acinus) Inducing Apoptotic Chromatin Condensation from Bovine Thymus Lysate
A factor inducing apoptotic chromatin condensation from bovine thymus lysate was purified in the same manner as in Example 1 described above by using this in vitro apoptosis assay.
All procedures described below were carried out at 0° to 4° C. Bovine thymus was homogenized in twice the volume of the lysis buffer. After centrifugation, the resulting supernatant was dialyzed against a buffer (20 mM Tris-HCl, pH 8.8, 2 mM MgCl2, 2 mM DTT and 0.5 mM EGTA), and the resulting dialysate was applied to a HiTrap Q column (5 ml×20) equilibrated with buffer A (20 mM Tris-HCl, pH 8.5, 2 mM MgCl2, 2 mM DTT, 0.5 mM EGTA, 2 mM β-glycerophosphate, and 250 mM sucrose). The column was washed with three times the volume of the column of buffer A, and proteins were eluted with 400 ml of a linear NaCl gradient (0–0.2 M). The resulting fractions of 8 ml were collected, and assayed for chromatin-condensing activity for its appropriate amount.
Since the bovine thymocytes used were partly caused to be apoptotic, inducers of apoptotic nuclear changes were expected to exist in the lysate as either proforms or active forms. Therefore, the assay was carried out in the presence of active caspase-3 in order to convert any proforms to their active forms. Components essential for active nuclear transport were supplemented to support the entry of proteins into the nucleus. After the bovine lysate was subjected to HiTrap Q column chromatography, three fractions, peaks A, B, and C, that induced chromatin condensation were detected, and peak A fraction caused typical apoptotic chromatin condensation without inducing DNA fragmentation in the oligonucleotide units. The results are shown in FIG. 2.
The factor in peak B was identified as the proform of caspase-6, a protease cleaving lamin A, by microsequencing the purified protein (data not shown), and the factor in peak C was identified as a CAD/ICAD (DFF40/45)-like DNase complex that induced DNA fragmentation in the oligonucleotide units after cleavage by caspase-3 (FIG. 2).
The Peak A fractions obtained by HiTrap Q column chromatography were passed through a hydroxyapatite column (5×10 cm) and were applied to a Heparin Sepharose column (1.5×3 cm). The column was washed with 50 ml of buffer B (composition: 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 2 mM DTT, 0.5 mM EGTA, 2 mM p-glycerophosphate, and 250 mM sucrose), and bound proteins were eluted with 10 ml of buffer B containing 1 M ammonium sulfate. The active fractions were applied to a Phenyl Sepharose column (0.8×2 cm), and the flow through fraction (10 ml) was supplemented with bovine serum albumin and concentrated to 0.5 ml. The sample was then loaded onto a Superose 12 column in buffer B. After the elution was carried out, the fractions obtained with activity were diluted ten-fold with buffer A and then applied to a Mono Q column equilibrated with buffer B. The column was washed with 20 ml of buffer A, and the elution was carried out with 30 ml of a linear NaCl concentration gradient (0–50 mM). Fractions (1 ml) were collected and stored at −80° C. Each purification step of p 17 is shown in FIG. 3 and Table 1.







TABLE 1







Purification of Acinus













Amount of
Entire
Specific
Purifica-



Purification
Proteina
Activityb
Activity
tion Ratio
Yield


Steps
(mg)
(U)
(U/mg)
(folds)
(%)















100000 × g
3680
ND
ND
ND
ND


(Supernatant)


HiTrap Q
186
14400
77.4
1
100


Hydroxy-
2.8
4000
1428
18.4
27.8


apatite/Heparin


Sepharose


Phenyl
0.5
1680
3368
43.4
11.7


Sepharose


Superose 12
0.1
1600
16000
207
11.1


Mono Q
0.001
1120
1120000
14470
7.78





aThe amount of protein was determined by using DC protein assay kit (manufactured by BioRad), and the amount of protein of Mono Q pool was deduced by silver staining of the gel after SDS-PAGE.


b1 U is defined as an activity for inducing nuclear condensation in 50% of the cells. In the table, ND means that the nuclear morphologies could not be determined owing to the existence of other factor.






As a result, a protein of about 17 kDa (p17) inducing chromatin condensation was purified (FIG. 3). In addition, as shown in FIG. 4, the purified protein induced apoptotic chromatin condensation in the absence of caspase-3, suggesting that this 17 kDa protein is an active form. This factor causing the chromatin condensation was named Acinus (Apoptotic chromatin condensation inducer in the nucleus).
EXAMPLE 3
Amino Acid Sequence Analysis of Acinus
The purified protein (about 2 μg) derived from the Mono Q column obtained in the manner as described in Example 2 was subjected to 15% SDS-PAGE, and thereafter electroblotting was carried out on Immobilon membrane (manufactured by Amersham). The membrane was stained with PonceusS, and thereafter a band of a size of 17 kDa was cut out. After subjecting the band to carboxymethylation as described in Matsudaira [Matsudaira, P., Academic Press, San Diego, 1993], the modified protein was digested with 1 pmole of Acromobacter protease I, and the resulting peptides were purified using mPRC C2/C18 SC 2.1/10 with a Smart System (manufactured by Pharmacia). The sequences of the four isolated peptides were determined by a protein sequencer manufactured by Applied Biosystems (Model 470A). The results are shown in FIG. 5.
As shown by the underlined portions in FIG. 5, the four internal peptide sequences derived from the active bovine Acinus corresponded to the deduced amino acid sequences of a human KIAA clone (KIAA0670) [Ishikawa, K. et al., DNA Research 5, 196–176 (1998)] in the database. Therefore, KIAA0670 seemed to represent a human autholog of bovine acinus.
EXAMPLE 4
1) cDNA Cloning Encoding Acinus
Based on the amino acid sequences of the four peptides obtained from the purified bovine Acinus fragment of a size of 17 kDa, it was identified that the four peptides corresponded to the KIAA clone (KIAA0670) in the BLAST database. KIAA0670 DNA was a kind gift from Dr. Ohara.
As a result of screening using KLAA0670 and the like, three isoforms of human acinus cDNA (the L, S and S′ forms), which were probably generated by differential splicing, were obtained. Concretely, the sequence corresponding to the 5′ end of acinusL was obtained from the above-mentioned KIAA0670 by using the RACE protocol of marathon-ready cDNA. acinusS′ cDNA was isolated by screening a cDNA library using PCR products corresponding to 3287–3669 bp of acinusL. acinusS cDNA was obtained by RT-PCR as a differential splicing isoform with an insertion upstream of the start codon of acinusS′. The acinus L, S, and S′ cDNA contained open reading frames of 1341 amino acid residues, 583 amino acid residues, and 568 amino acid residues, respectively (see FIGS. 5 and 6).
Further, mouse acinus cDNAs were obtained by screening a library with human acinus cDNA. The mouse Acinus proteins corresponding to the human AcinusS and S′ showed 94.5% and 95.2% homology of the amino acid sequences with the human AcinusS and S′, respectively. A protein database (BLAST) search revealed that Acinus derived from human or mouse contained a region resembling the RNA recognition motif of Drosophila S×1. The results are shown in FIG. 7. Only AcinusL had a P-loop for nucleotide binding near the N-terminus.
2) Preparation of Acinus Recombinant Protein (rAcinus)
Full length rAcinusS and rAcinusΔN were produced as a GST fusion protein and a His6-tagged protein, respectively.
Concretely, acinusS cDNA was subcloned into pGEX1T (manufactured by Pharmacia), and thereafter E. coli DHa strain was transfected by using the resulting plasmid. Protein production was induced by the addition of IPTG (final concentration: 0.1 mM). Bacteria were incubated at 20° C. overnight and lysed in the same lysis buffer as that used for the protein derived from bovine thymus. GST-AcinusS was purified with Glutathione Sepharose CL-4B (Pharmacia) according to the protocol of the manufacturer. The purity was estimated to be about 90% by CBB staining.
acinusΔN and acinusΔN (D/A) (one resulting from substitution of 1093rd aspartic acid with alanine) cDNA were subcloned into pREST (manufactured by Invitrogen). Expression of the protein was induced by infection with M13 phage for producing T7 polymerase (M13-KM2), and His-tagged Acinus proteins were purified on nickel affinity column (manufactured by Novagen).
rAcinus (987–1093) was prepared by cleavage of rAcinusΔN with caspase-3 and was purified by HiTrap Q column chromatography. Since the purity of the His-tagged proteins was not high (purity of about 10%), the amount of the protein was estimated from the concentration visualized by immunoblotting.
The apparent molecular weights of AcinusL, S, and S′ on SDS-polyacrylamide gel were about 220 kDa, about 98 kDa and about 94 kDa, respectively (data not shown). The human AcinusL, S, and S′ along with low-molecular weight Acinus were present in Jurkat cells (FIG. 8). The majority of the three forms of the human Acinus was recovered in a TritonX-100-insobuble fraction, while most of the low-molecular weight Acinus was a soluble fraction (FIG. 8). The nature of low-molecular weight Acinus was not determined, and they might have been in proteolytically processed forms or unidentified isoforms of Acinus. During Fas-mediated apoptosis, as shown in FIG. 8, AcinusL, S, and S′ as well as some of the low-molecular weight Acinus were reduced before chromatin condensation became evident, while an 85 kDa protein in the insoluble fraction and a 23 kDa protein in the soluble fraction newly emerged accompanying chromatin condensation. This suggests that Acinus was cleaved by the caspase, generating active fragments.
EXAMPLE 5
Identification of Active Form of Human Acinus
Purified active bovine Acinus p17 seemed to be subjected to truncation at both N- and C-terminal sites, on the bases that (1) N-terminal sequence analysis revealed that p17 started as Ser987, and (2) the size of p17 indicated C-terminal truncation. A plural forms of recombinant Acinus (hereinafter referred to as “rAcinus”): AcinusS, ΔN (from Ser987 to the C-terminus), ΔN (D/A), and Acinus (987–1093) (from Ser987 to Asp1093) were prepared, and their chromatin-condensing activity, cleavage by caspase-3, and the role of N-terminal site in activation of Acinus were studied.
As shown in FIG. 9, human AcinusS was actually cleaved with caspase-3 in vivo, and the cleavage site was determined to be the Asp1093 of DELD1093 by microsequencing of the cleavage product of recombinant AcinusS. This result was identical to the consensus DxxD target sequence for caspase-3. Consistently, a mutation of substituting Asp1093 with Ala (D/A) abolished the in vitro cleavage of Acinus by caspase-3.
In addition, as shown in FIG. 10, rAcinusΔN exhibited chromatin-condensing activity in the presence of caspase-3, but did not exhibit chromatin-condensing activity in the presence of a caspase-3 inhibitor DEVD-CHO or in the absence of caspase-3, and at the same time rAcinusΔN with Asp1093/Ala (D/A) mutation [ΔN(D/A)] did not induce chromatin condensation in the presence of caspase-3. These results indicated that cleavage of rAcinusΔN by caspase-3 at Asp1093 was essential for the chromatin-condensing activity.
In addition, as shown in FIG. 10, rAcinusΔN preincubated with caspase-3 induced chromatin condensation even in the presence of DEVD-CHO, indicating that caspase-3 activity was no longer required for chromatin condensation after cleavage of Acinus. Supporting this fact, rAcinus (987–1093) induced chromatin condensation in the absence of caspase-3 and in the presence of DEVD-CHO, similarly to bovine Acinus p17.
Further, as shown in FIG. 10, although the possibility that rAcinusS was not properly folded was not excluded, since full length rAcinusS had no chromatin-condensing activity, it is seen that cleavage at Ser987 of the full length rAcinusS was also necessary for chromatin activity. The results for the observation (FIG. 8) that apoptotic Jurkat cells contained a 23 kDa protein with a size corresponding to rAcinus (987–1093) in a newly emerging Acinus fragment indicated that Acinus (987–1093) was one of the active forms of Acinus which induced chromatin condensation. Therefore, it is evident that the active form of bovine Acinus p17 corresponded to Acinus (987–1093), and the size difference between human and bovine active Acinus might have been due to some amino acid changes or further truncation at the C-terminus of bovine Acinus.
Active nuclear transport proteins were required for the induction of chromatin condensation by rAcinus (FIG. 10), suggesting that nuclear localization of Acinus was essential for its chromatin-condensing activity in the in vitro apoptosis assay system. In fact, a significant amount of rAcinusΔN was translocated to the nucleus in vitro as shown by immunostaining of permeabilized cells. In addition, as shown in FIG. 11, since rAcinusΔN (D/A) was also transported to the nucleus, cleavage of Acinus by caspase was not essential for nuclear localization. As shown in FIG. 11, endogenous Acinus was mainly detected in the nucleus, suggesting a possibility that the caspase is transported into the nucleus to proteolytically activate Acinus during in vivo apoptosis. Alternatively, the chromatin condensation is thought to be induced after a small amount of Acinus or proteolytically processed Acinus may be present in the cytoplasm, cleaved by the caspase and transported to the nucleus. Localization of the endogenous Acinus in discrete areas of the nucleus suggested its association with a certain kind of a nuclear structure.
Consistent with the observation that purified bovine Acinus caused chromatin condensation without oligonucleosomal DNA fragmentation, as shown in FIG. 12, no DNA fragmentation was observed when rAcinus induced chromatin condensation in vitro. Therefore, it is suggested that Acinus produces apoptotic chromatin condensation by a mechanism independent of DNA fragmentation in the oligonucleotide units.
EXAMPLE 6
Influence of Immunodepletion of Acinus
In order to determine whether or not Acinus was essential for apoptotic chromatin condensation, the ability of inducing chromatin condensation in vitro of normal Jurkat cell lysate and apoptotic Jurkat cell lysate each immunodepleted of Acinus was tested. Jurkat cell lysate was prepared as described above. Antibody-loaded protein G-beads were prepared as described in Hirano et al. [Hirano, T. et al., Cell 89, 511–521 (1997)]. For immunodepletion of Acinus, lysates were incubated with an equal volume of the above beads at 4° C. for 2 hours with rotation. The supernatants were recovered by two rounds of brief spinning to be used as immunodepleted lysates.
The reduction in the amount of Acinus in the normal cell lysate and the apoptotic cell lysate was confirmed by immunoblotting (FIG. 13), whereas CAD/DFF40 activity as evaluated with the permeabilized cells remained unchanged. Although it is seen that the immunoblot profile shown in FIG. 13 is slightly different from one in FIG. 8, it is probably due to a much larger number of cells used to prepare the lysates in FIG. 13. As shown in FIG. 14, lysates immunodepleted with anti-Acinus antibody did not induce chromatin condensation. Addition of rAcinusΔN restored the chromatin-condensing activity of the immunodepleted lysates. Also, the lysate treated with a control antibody showed chromatin-condensing activity. These results indicated that Acinus was indispensable for apoptotic chromatin condensation in the in vitro system. The lysates immunodepleted with the anti-Acinus antibody, as shown in FIG. 14, caused slight accumulation of chromatin at the nuclear periphery and slight nuclear shrinkage, which may have been due to the incomplete immunodepletion of Acinus or due to the presence of other factors causing nuclear changes, including caspase-6 and CAD/DFF40.
EXAMPLE 7
Transfection and Analysis of Nuclear Changes
The role of Acinus in the in vivo apoptotic process was examined by transient transfection of sense or antisense acinusL cDNA into HeLa cells.
acinusL, S, S′, and S′(D/A) cDNAs were cloned in the pCAGGS vector [Niwa, H. et al., Gene 108, 193–199 (1991)]. Further, acinusL cDNA was cloned in the vector in an opposite orientation [acinusL(R)]. acinus cDNA (1 μg) was transfected into 0.5×106 HeLa or COS-7 cells together with 0.1 μg of pEGFP-C1 (manufactured by Clontech) using Lipofectamine (manufactured by Gibco). Cells were incubated for 48 hours in medium supplemented with 10% fetal bovine serum. For immunoblotting, cells were lysed with SDS sample buffer and subjected to SDS-PAGE. Cell death was induced by the addition of 100 μM etoposide. After the indicated period, cells were stained with Hoechst 33342. Subsequently, the GFP-positive apoptotic cells and live cells were counted.
A GFP marker plasmid was co-transfected to identify the DNA-transfected cells. The sense plasmid caused slight induction of chromatin condensation without any apoptotic stimuli, and enhanced apoptotic chromatin condensation induced by etoposide, topoisomerase II inhibitor (FIG. 15). Conversely, in the antisense plasmid, chromatin condensation induced by etoposide was effectively delayed (FIG. 15). After transfection with the antisense acinus plasmid, down-regulation specific to endogenous Acinus was verified by immunoblotting (FIG. 16). Further, expression of Acinus (987–1093) in HeLa cells, which was active in inducing chromatin condensation in vitro, was also tried, but its expression was undetectable, probably due to instability in vivo. These results suggested that Acinus further played a significant role in apoptotic chromatin condensation in vivo.
It has been reported that CAD/DFF40 can induce chromatin condensation in isolated nuclei, and that in thymocytes and splenocytes being deficient of DFF40 (consequently being deficient of functional DFF40), apoptotic chromatin condensation is impaired, suggesting that CAD/DFF40 is involved in chromatin condensation. However, there may be other factors required for apoptotic chromatin condensation, because (1) apoptotic chromatin condensation is not completely abolished in DFF40-deficient cells, (2) ICAD (an inhibitor of CAD/DFF40) does not inhibit chromatin condensation is isolated nuclei induced by apoptotic cell lysates, and (3) CAD is not expressed in human tissues and cell lines where apoptotic chromatin condensation is observed. Since Acinus is ubiquitously expressed, Acinus is likely to play an important role in apoptotic chromatin condensation in general, even though CAD/DFF40 might play a dominant role in inducing chromatin condensation in certain cells. It is also possible that Acinus-induced chromatin condensation is facilitated by CAD/DFF40.
Acinus is essential for apoptotic chromatin condensation, and it might also be involved in nuclear structural changes occurring in normal cells, such as chromatin condensation in the M-phase and nuclear mitosis. Acinus has a P-loop motif and a region similar to the RNA recognition motif of S×1, suggesting that Acinus might possess ATPase activity and DNA/RNA binding activity. Further studies on Acinus are considered to serve to elucidate not only the process of apoptotic nuclear changes but also nuclear functions in viable cells.
Sequence Listing Free Text
In SEQ ID NO: 5, n in the base number: 1 means that an exact sequence could not be determined because the signals overlapped.
In SEQ ID NO: 6, each of n in the base numbers: 20, 104, 114, 148 and 156 means that an exact sequence could not be determined because the signals overlapped.
INDUSTRIAL APPLICABILITY
According to the polypeptide, the nucleic acid, and the antibody of the present invention, there can be exhibited excellent effects such that the polypeptide, the nucleic acid, and the antibody can be utilized for searches for a factor for which the polypeptide is used as a target molecule in control of apoptosis, searches for a molecule for which the polypeptide is targeted, and the like, and further that they can be effectively used for controlling apoptosis. In addition, according to the agent for controlling apoptosis and the method of controlling apoptosis, the apoptosis can be positively or negatively controlled. In the agent for controlling apoptosis and the method of controlling apoptosis mentioned above, their applications to various diseases accompanying apoptosis are expected.

## Claims

1. A polypeptide possessing an action of causing in vitro chromatin condensation in the presence of caspase-3, consisting of a sequence selected from the group consisting of:
(A) the amino acid sequence of SEQ ID NO:4; and
(B) an amino acid sequence encoded by a nucleic acid consisting of 321 nucleotides that hybridizes to the complete complement of SEQ ID NO:8, wherein said hybridizing occurs at 42° C. in a solution containing 6×SSC, 0.5% SDS and 50% formamide solution, with washing thereafter at 68° C. in a solution containing 0.1×SSC and 0.5% SDS, wherein said amino acid encoded by the nucleic acid possesses an action of causing in vitro chromatin condensation in the presence of caspase-3.

2. The polypeptide according to claim 1, wherein the polypeptide possesses an action of causing in vitro chromatin condensation without accompanying DNA fragmentation.

3. An agent for causing in vitro chromatin condensation, comprising the polypeptide of claim 1 or 2.

4. A screening method for a test substance for controlling chromatin-condensing activity in the presence of caspase-3, comprising
(a) incubating the test substance with permeabilized cells in the presence of the polypeptide of claim 1 or 2; and
(b) evaluating an activity of causing chromatin condensation exhibited by the polypeptide.

5. A screening method for a test substance for controlling chromatin-condensing activity in the presence of caspase-3, comprising
(a) incubating the test substance with permeabilized cells in the presence of the polypeptide of claim 1 or 2; and
(b) evaluating an activity of inhibiting chromatin condensation exhibited by the polypeptide.

6. The screening method according to claim 4, wherein said activity is enhancing chromatin condensation.

7. The polypeptide according to claim 1, wherein said polypeptide is encoded by a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 8.

8. A polypeptide for causing in vitro chromatin condensation, consisting of the polypeptide of claim 1 or 2.