JP5891561B2 - Mitochondrial membrane proteins and genes encoding them - Google Patents

Description

  The present invention relates to a protein having a function of controlling or regulating mitochondrial morphology, and a gene encoding the protein. The present invention also provides a novel protein having a function of controlling or regulating the form of mitochondria, which comprises exhaustively purifying and isolating a protein present in mitochondria, preferably a mitochondrial membrane protein, and encodes the protein. Related to the gene. Furthermore, the present invention provides an activity to normalize the mitochondrial morphology using cells in which a protein having a function of controlling or regulating the mitochondrial morphology is deficient or excessive and the mitochondrial morphology is abnormal. The present invention relates to a method for screening a substance having the same.

Mitochondria are eukaryotic organelles, and it is thought that aerobic prokaryotic cells are symbiotic with primitive eukaryotic cells. Mitochondria have its own DNA separate from the cell nucleus, which is called mitochondrial DNA (mtDNA). While mtDNA is relatively constant at 16-18 kb in animals, it is extremely diverse at 6-77 kb in protozoa. So far, the entire base sequence of mtDNA of nearly 700 species has been analyzed, and the genes common to all mtDNAs are two rRNAs and two genes involved in the electron transport system (cob, And cox1). Mammalian mtDNA such as human is a circular double-stranded DNA of about 16.5 kb, and several hundred to several thousand copies are present per cell. Unlike nuclear DNA, mtDNA encodes genes with almost no gap, and humans encode 13 types of proteins, 2 rRNAs, and 22 types of tRNAs.
Mitochondria are said to be composed of about 1500 proteins in mammals. Since only 13 of these proteins are produced by mtDNA, the remaining approximately 99% of the protein is encoded by the DNA of the cell nucleus. Proteins synthesized in the cytoplasm are selectively transported to the mitochondria by the mitochondrial directing sequence (MTS) present at the N-terminus. A method of supplementing mitochondrial proteins with such a signal sequence has also been developed (see Patent Documents 1 and 2).

  Mitochondria produce most of the ATP necessary for life activity. It also has important functions such as induction of apoptosis and generation of active oxygen. These functions depend on the action of the electron transport system existing on the inner mitochondrial membrane. The mitochondrial membrane is composed of two membranes, an outer membrane and an inner membrane, and the inner membrane has a finger-like structure called christe. The form of this crystal depends on the mitochondrial energy state. Compared to the resting state, it is known that the intermembrane space expands and the structure of the crystal is dense in the high energy state, and the mitochondrial membrane form is the function of the electron transport system. It seems to be closely related to regulation. So far, it has been observed that the morphology of eukaryotic mitochondria changes dynamically with cell differentiation and pathological state, especially based on liver disease, congenital muscular dystrophy, gastric cancer and myeloma, and mitochondrial DNA abnormalities It is known that mitochondrial morphology and distribution changes significantly in pathological conditions such as dilated cardiomyopathy, and presents mitochondria, circular or axle-like mitochondria, mitochondria with circular or concentric criste structure, etc. (See Non-Patent Documents 1 and 2). Thus, it is considered that cell functions and mitochondrial morphology are closely related, and mitochondrial morphology abnormalities have been reported even in diseases not related to mitochondrial DNA mutation (see Non-Patent Documents 3, 4 and 5). ). In addition, encephalomyopathy (see Non-Patent Document 6), Alzheimer's disease and Parkinson's disease (see Patent Documents 3 and 4, and Non-Patent Documents 7 and 8) are also associated with mitochondrial abnormalities, and type II It has also been reported that diabetes occurs in mitochondrial tRNA having at least a 3243A-G mutation (see Non-Patent Document 9). And the pharmaceutical composition for treating such various mitochondrial diseases has also been developed (refer patent documents 5 and 6).

On the other hand, lifestyle-related diseases and aging-related diseases in an aging society are important. It is known that the function of mitochondria is closely related to lifestyle-related diseases, aging, and various neurological diseases. For example, it is known that mitochondria produce ATP and consume a large amount of oxygen, but 0.4 to 4% of oxygen consumed by mitochondria is not related to ATP production and becomes active oxygen. It is thought that this reactive oxygen produced in mitochondria damages cells, reduces the number of cells, and promotes the deterioration of cell functions and aging of individuals.
In addition, abnormalities in mitochondrial morphology have been reported in cancer and nutritional disorders. By using a gene fragment encoding a protein group revealed by the present invention, an antibody recognizing a protein, etc., it is expected to be applied to a disease diagnosis marker in the future. These proteins can also be considered as therapeutic targets, and these protein derivatives can also be applied to therapeutic drugs.

Therefore, maintaining mitochondrial functions normally and controlling them well is not only important for preventing aging but also maintaining individual homeostasis.
However, it is said that there are about 1500 kinds of proteins in mitochondria, but what kind of proteins exist, what functions these proteins have, and what relationships they have. It has not been clarified yet. In addition, the detailed molecular mechanism that regulates the mitochondrial membrane morphology and the link between mitochondrial membrane morphology and function have not yet been clarified.
Many studies have been made in recent years in order to elucidate various functions of mitochondria. For example, those related to ion permeability of mitochondrial membrane (see Patent Document 7), those related to caspase activation (see Patent Document 8), those related to intracellular movement of proteins into mitochondria (see Patent Document 9), mitochondrial DNA A method related to the gene detection method used (see Patent Document 10) has been reported.

JP 2001-224389 A JP 10-146200 A JP 2005-82523 A JP-A-2005-151810 JP 2002-523434 A JP 2004-538326 A JP 2004-189730 A JP 2004-67531 A JP 2004-41067 A JP 2003-116576 A

Arbustini E, Diegoli M, et al., Am J Pathol. 1998 Nov; 153 (5): 1501-10. Nishino I, Kobayashi O, et al., Muscle Nerve. 1998 Jan; 21 (1): 40-7. Ann. NY Acad. Sci., 488, 65-81, 1986 Acta haemat. 68, 241-249, 1982. Scand. J. Gastroenterol. 33, 975-981, 1998. Goto, Y .; Nonaka, I .; et al., Nature 348: 651-653, 1990. Lustbader, J.W., Cirilli, M., Et al., Science 304 (5669), 448-452 (2004) Shoffner, J. M., et al., Genomics 17: 171-184, 1993. Ouweland, J. M. W .; Lemkes, H. H. P. J .; et al., Nature Genet. 1: 368-371, 1992.

In this way, the function of mitochondria is extremely important in maintaining the homeostasis of an individual, and it is necessary to individually analyze a wide variety of functions of mitochondria, but comprehensively cover the proteins responsible for the function of mitochondria Therefore, it is desired to elucidate the functions and mutual relationships of proteins that regulate and analyze mitochondrial membrane morphology and regulate mitochondrial function.
The present invention comprehensively identifies and analyzes proteins responsible for mitochondrial functions, and elucidates the functions of proteins and their relevance that regulate mitochondrial membrane morphology and regulate mitochondrial functions. The present invention also provides a novel protein identified and analyzed by the method of the present invention and its use, and further provides a method for regulating mitochondrial function.

The present inventor studied a method for easily and efficiently isolating and purifying mitochondria, and then examining a method for separating a mitochondrial membrane fraction and a matrix fraction from the obtained mitochondrial fraction. A method for separating proteins in the membrane fraction into two protein groups, a protein group having a property of binding tightly to a membrane such as having a transmembrane domain, and a protein group loosely binding to the membrane was examined. As a result, the present inventor separated the crude mitochondrial fraction by centrifugation after cell disruption from human cultured cells cultured in large quantities, and purified this fraction by specific gravity density gradient centrifugation. It was found that a highly pure mitochondrial fraction can be obtained. Then, this purified mitochondrial fraction is sonicated and separated into a membrane fraction and a matrix fraction by centrifugation, and the obtained membrane fraction is washed with an alkaline solution to loosen the membrane. Two protein groups of the protein group to be bound were separated, and it was found that a protein group having a property of firmly binding to a membrane such as having a transmembrane domain can be obtained from the remaining membrane fraction.
Then, the present inventor has found that the protein group that strongly binds to the mitochondrial membrane can be solubilized, and these proteins can be comprehensively analyzed by two-dimensional electrophoresis. Furthermore, the obtained protein spots were separated, these spots were cut out, peptides were extracted after in-gel digestion, and these proteins were successfully identified by mass spectrometry and database search.

That is, the present invention has an amino acid sequence described in any one of SEQ ID NOs: 1 to 15 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue It relates to a protein having More specifically, the present invention has an amino acid sequence described in any of SEQ ID NOs: 1 to 12 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and a mitochondrial membrane tissue It relates to a protein having the ability to form
The present invention also has an amino acid sequence set forth in any one of SEQ ID NOs: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue Tested on cells in which one or more proteins selected from the group of proteins having deficiency are deficient or overexpressed, or cells to which these proteins have been added and in which mitochondrial morphology is abnormal The present invention relates to a method for screening an active substance for normalizing mitochondrial morphology, comprising adding a substance and measuring a change in mitochondrial morphology by the test substance.
Furthermore, the present invention has an amino acid sequence described in any one of SEQ ID NOs: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue The present invention relates to a mitochondrial membrane tissue-forming agent comprising a protein having The present invention also has an amino acid sequence set forth in any one of SEQ ID NOs: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue It relates to a mitochondrial membrane shape regulator comprising a protein having Furthermore, the present invention has an amino acid sequence described in any one of SEQ ID NOs: 1 to 12 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue And a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
The present invention also provides a mitochondrial membrane tissue using an oligonucleotide consisting of a base sequence set forth in any one of SEQ ID NOs: 23 to 44 in the sequence listing, or a sequence of at least 20 consecutive bases of these base sequences. The present invention relates to a method for detecting and identifying a gene encoding a protein to be formed.
Furthermore, the present invention has an amino acid sequence described in any one of SEQ ID NOs: 1 to 15 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and the ability to form mitochondrial membrane tissue It relates to an antibody against a protein having

In addition, the present invention separates a crude mitochondrial fraction from cultured cells after cell disruption by centrifugation, and purifies this fraction by specific gravity gradient centrifugation, thereby obtaining a high-purity mitochondrial fraction. It relates to a method of manufacturing. More specifically, in the present invention, the purified mitochondrial fraction is sonicated and then separated into a membrane fraction and a matrix fraction by centrifugation, and the obtained membrane fraction is washed with an alkaline solution. Mitochondria consisting of separating the two protein groups of the protein group that binds loosely to the membrane, and obtaining from the remaining membrane fraction a protein group that has a property of firmly binding to the membrane, such as having a transmembrane domain The present invention relates to a method for producing a protein group forming a membrane tissue. More specifically, the present invention separates a crude mitochondrial fraction from a cultured cell after cell disruption by a centrifugation method, and purifies this fraction by a specific gravity gradient centrifugation method. The mitochondrial fraction is obtained, and then the purified mitochondrial fraction obtained is sonicated and then separated into a membrane fraction and a matrix fraction by centrifugation, and the obtained membrane fraction is washed with an alkaline solution. By separating the two protein groups of the protein group that binds loosely to the membrane, and obtaining the protein group having the property of firmly binding to the membrane, such as having a transmembrane domain, from the remaining membrane fraction The present invention relates to a method for producing a protein group forming a mitochondrial membrane tissue.
The present invention also relates to a method for solubilizing a sample containing a protein group forming a mitochondrial membrane tissue and comprehensively analyzing these protein group by two-dimensional electrophoresis.
Furthermore, the present invention is to solubilize a sample containing a protein group forming a mitochondrial membrane tissue, obtain a migration pattern by two-dimensional electrophoresis of these protein group by two-dimensional electrophoresis, and obtain the obtained electrophoresis The present invention relates to a method for detecting and identifying an abnormality in a protein group forming a mitochondrial membrane tissue comprising comparing a pattern with a standard migration pattern.

Describing the present invention in more detail, the present invention is as described in the following (1) to (28).
(1) A protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 15 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and having the ability to form mitochondrial membrane tissue .
(2) A protein having the amino acid sequence of any one of SEQ ID NOs: 1 to 12 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and having the ability to form mitochondrial membrane tissue .
(3) The protein according to (1) or (2) above, wherein the protein is a protein localized in mitochondria.
(4) The protein according to (1) or (2) above, wherein the protein has a function of controlling or regulating mitochondrial morphology.
(5) The protein is obtained by crushing cultured cells, separating a crude mitochondrial fraction by centrifugation, and purifying the fraction by specific gravity density gradient centrifugation (see above). The protein according to 1) or (2).
(6) A protein having the amino acid sequence set forth in any one of SEQ ID NOS: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and having the ability to form mitochondrial membrane tissue A test substance is added to cells in which one or more proteins selected from the group described above are deficient or overexpressed, or cells to which these proteins have been added, in which abnormal mitochondrial morphology has occurred A method of screening for a substance active to normalize the mitochondrial morphology, comprising measuring changes in mitochondrial morphology by the test substance.
(7) The protein group has an amino acid sequence according to any one of SEQ ID NOs: 1 to 15 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, The method according to (6) above, which is a group of proteins having forming ability.
(8) The protein group has an amino acid sequence described in any one of SEQ ID NOS: 19 to 22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, The method according to (6) above, which is a group of proteins having forming ability.
(9) The method according to any one of (6) to (8), wherein the normalization of mitochondrial morphology is suppression of mitochondrial aggregation.
(10) A protein having an amino acid sequence described in any one of SEQ ID NOS: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and having the ability to form a mitochondrial membrane tissue A mitochondrial membrane tissue-forming agent.
(11) A protein having the amino acid sequence of any one of SEQ ID NOS: 1-22 in the sequence listing, or an amino acid sequence having at least 80% homology with these amino acid sequences, and having the ability to form mitochondrial membrane tissue A mitochondrial membrane shape regulator comprising:
(12) It has an amino acid sequence described in SEQ ID NO: 1 to 15, preferably SEQ ID NO: 1 to 12, or an amino acid sequence having at least 80% homology with these amino acid sequences, and mitochondria A pharmaceutical composition comprising a protein capable of forming a membrane tissue and a pharmaceutically acceptable carrier.
(13) A protein that forms a mitochondrial membrane tissue using an oligonucleotide consisting of the base sequence set forth in any one of SEQ ID NOs: 23 to 44 in the sequence listing or a continuous sequence of at least 20 bases of these base sequences A method for detecting and identifying a gene coding for.
(14) It has an amino acid sequence described in SEQ ID NO: 1 to 15, preferably any of SEQ ID NO: 1 to 12, or an amino acid sequence having at least 80% homology with these amino acid sequences, and mitochondria An antibody against a protein capable of forming a membrane tissue.
(15) The antibody according to (14), wherein the antibody is a monoclonal antibody.

(16) After cell disruption, the crude mitochondrial fraction is separated from the cultured cells by centrifugation, and the fraction is purified by specific gravity gradient centrifugation to produce a highly pure mitochondrial fraction. Method.
(17) The method according to (16) above, wherein the cultured cells are human cells.
(18) The method according to (17) above, wherein the human cell is a human HeLa cell.
(19) The purified mitochondrial fraction is sonicated and then separated into a membrane fraction and a matrix fraction by centrifugation, and the obtained membrane fraction is washed with an alkaline solution to loosen the membrane. A protein that forms a mitochondrial membrane tissue by separating two protein groups of the protein group that binds and obtaining a protein group that has a property of firmly binding to a membrane such as having a transmembrane domain from the remaining membrane fraction A method of manufacturing a group.
(20) The method according to (19) above, wherein the mitochondrial fraction is a mitochondrial fraction derived from human cells.
(21) From the cultured cells, after disrupting the cells, the crude mitochondrial fraction is separated by centrifugation, and the fraction is purified by specific gravity gradient centrifugation to obtain a highly pure mitochondrial fraction, The purified mitochondrial fraction obtained is then sonicated and then separated into a membrane fraction and a matrix fraction by centrifugation, and the membrane fraction obtained is washed with an alkaline solution to obtain a membrane. Separating two protein groups of loosely bound proteins, and forming a mitochondrial membrane tissue consisting of obtaining a protein group that has a property of firmly binding to a membrane such as having a transmembrane domain from the remaining membrane fraction A method for producing a protein group.
(22) The method according to (21), wherein the cultured cells are human cells.
(23) The method according to (22), wherein the human cell is a human HeLa cell.
(24) A method of solubilizing a sample containing a protein group forming a mitochondrial membrane tissue and comprehensively analyzing the protein group by two-dimensional electrophoresis.
(25) The method according to (24) above, wherein a sample containing a protein group forming a mitochondrial membrane tissue is obtained by the method according to any one of claims 4 to 9.
(26) A sample containing a protein group forming a mitochondrial membrane tissue is solubilized, and a migration pattern by two-dimensional electrophoresis of these protein group is obtained by two-dimensional electrophoresis, and the obtained migration pattern is standardized. A method for detecting and identifying an abnormality in a protein group forming a mitochondrial membrane tissue comprising comparing with an electrophoretic pattern.
(27) The method according to (26), wherein the sample containing a protein group that forms a mitochondrial membrane tissue is obtained by the method according to any one of claims 4 to 9.
(28) A sample containing a protein group forming a mitochondrial membrane tissue obtained from a cell having a normal mitochondrial function is solubilized and subjected to two-dimensional electrophoresis of these protein group by two-dimensional electrophoresis. Electrophoretic pattern.

Next, the present invention will be described in more detail.
The “mitochondrion” in the present invention may be from a biological species having mitochondria, and may be an animal or a plant. Preferred mitochondria include human mitochondria. By targeting human mitochondria, it can be applied to prevention, treatment or diagnosis of human diseases, health, aging, and the like.
The “mitochondrial membrane tissue” in the present invention refers to a group of substances, preferably a protein group, that binds strongly to the outer and / or inner membrane of mitochondria. A protein that forms a typical “mitochondrial membrane tissue” includes a protein having at least one transmembrane domain. For example, Tom40, Tom5, Tom6, Tom7, Tom22 that form a TOM40 complex that recognizes a mitochondrial localization signal in the outer membrane, and Tim17 that forms a TIM complex that forms a membrane permeation device in the inner membrane, Examples thereof include membrane proteins such as Tim22 and Tim23.
The “mitochondrial membrane tissue-forming agent” in the present invention is a substance for forming the “mitochondrial membrane tissue” of the present invention, preferably a protein. There are a large number of substances including known and unknown substances in the mitochondrial membrane tissue, and these are various, such as mitochondrial ATP production and apoptosis, alone or in combination or in relation to each other. It expresses various functions. Therefore, maintaining the “mitochondrial membrane tissue” normally is extremely important in maintaining the homeostasis of the individual. The “mitochondrial membrane tissue-forming agent” of the present invention is not only a substance functioning in normal mitochondrial membrane tissue, preferably a protein, but also an abnormal mitochondrial membrane that is lost or dysfunctional for some reason Includes what is applied to return the tissue to a normal state.
The “electrophoretic pattern” in the present invention is positional information based on a two-dimensional spot formed by spots formed by these protein groups when a solubilized protein group is subjected to two-dimensional electrophoresis. Such position information may be image information such as a photograph or video, or numerical information on the X axis and the Y axis. Therefore, the “electrophoretic pattern” in the present invention is not limited to a visually observable image, but can be said to be position information obtained by two-dimensional electrophoresis.
In addition, the development method in the two-dimensional electrophoresis of the present invention is preferably a development method based on a difference in molecular weight by the isoelectric focusing method in the first dimension and the SDS-PAGE method in the second dimension, but is not limited thereto. Absent. Various methods can be adopted as the first-dimensional and second-dimensional expansion methods. For example, a development method using an unmodified acrylamide gel often used for complex analysis or the like can also be adopted, but a combination of isoelectric focusing and SDS-PAGE is preferable because of good reproducibility.
The meaning of other terms in the present invention is used in the meaning normally used by those skilled in the art.

Next, the present invention will be described more specifically, but the present invention is not limited to these specific examples.
First, human cultured cells HeLa cells cultured in large quantities were disrupted, and the crude mitochondrial fraction was separated by centrifugation. A high-purity mitochondrial fraction was purified from this fraction by specific gravity density gradient centrifugation. The mitochondrial fraction was sonicated and then separated into a membrane fraction and a matrix fraction by centrifugation. This membrane fraction contains two protein groups, a protein group that has a property of binding tightly to the membrane, such as having a transmembrane domain, and a protein group that binds loosely to the membrane, and is washed with an alkaline solution. These were separated by In order to comprehensively analyze the protein group that strongly binds to the mitochondrial membrane, these proteins were first solubilized, and 200 or more protein spots were separated by two-dimensional electrophoresis. These spots were cut out, peptides were extracted after in-gel digestion, and these proteins were identified by mass spectrometry. A database search was performed on the identified proteins using peptide sequence tagging, and all proteins in the spots were identified. 247 spots were analyzed and 171 proteins were identified.
The protein group comprehensively identified and analyzed by the above method is considered to contain proteins that regulate mitochondrial membrane morphology and mitochondrial function.

A two-dimensional electrophoresis of the mitochondrial matrix fraction obtained by the above method is shown in FIG. Two-dimensional electrophoresis of the matrix fraction and the separated membrane fraction is shown in FIG. And the two-dimensional electrophoresis of the protein group which couple | bonds loosely with the film | membrane obtained by wash | cleaning a membrane fraction with an alkaline solution is shown in FIG. FIG. 4 shows two-dimensional electrophoresis of a protein group having a property of firmly binding to a membrane such as having a transmembrane domain. The vertical direction of the two-dimensional electrophoresis photographs of FIGS. 1 to 4 indicates the molecular weight, and the horizontal direction indicates the pH.
The patterns developed by these two-dimensional electrophoresis contained phosphorylated proteins and various isoforms.
By this method, 247 spots of proteins forming mitochondrial membrane tissue were analyzed, and 171 proteins excluding duplication were identified. The results of classifying these 171 proteins based on mitochondrial function are shown in Table 1 below.

As a result of identifying these 171 types of proteins, the following 22 types of proteins were obtained as proteins other than proteins whose functions were identified, such as enzymes, skeletons, chaperones, and transporters. The amino acid sequences of these proteins are listed in 1-22 of the sequence listing, respectively. In addition, the nucleotide sequences encoding these proteins are described in 23 to 44 of the sequence listing, respectively.
When the database was searched based on the amino acid sequence of the obtained protein, it could be divided into the following three groups A to C.
Group A: Proteins whose sequences have been estimated in genomic studies, but have not been isolated and confirmed so far, and their functions have not been clarified.
The protein set forth in SEQ ID NO: 1 NP-060336 (LOC54968)
hypothetical protein LOC54968
Protein described in SEQ ID NO: 2: NP-077027 (LOC79135)
hypothetical protein LOC79135
Protein described in SEQ ID NO: 3: NP-001001692 (FLJ45139)
FLJ45139 protein
Protein described in SEQ ID NO: 4: NP-775932 (LOC285492)
hypothetical protein LOC285492
The protein of SEQ ID NO: 5: NP-078937 (LOC79714)
hypothetical protein LOC79714
Protein described in SEQ ID NO: 6: AAQ89151 (AAQ89151)
HSAL5836
Protein described in SEQ ID NO: 7: NP-115716 (c6orf125)
chromosome 6 open reading frame 125
The protein described in SEQ ID NO: 8: BAC04486 (BAC04486)
unnamed protein product
The protein set forth in SEQ ID NO: 9: NP-951032 (E3-3a)
nuclear protein E3-3 isoform a
The protein described in SEQ ID NO: 10: NP-060282 (CHCHD3)
coiled-coil-helix-coiled-coil-helix domain
containing 3; CHCHD3
The protein described in SEQ ID NO: 11: NP-057026 (CGI-12)
hypothetical protein LOC51001; CGI-12
Protein described in SEQ ID NO: 12: Q9H078 (Q9H078SKD3)
suppressor of potassium transport defect 3; SKD3
Protein described in SEQ ID NO: 13: NP-060300 (OCIA)
ovarian carcinoma immunoreactive antigen; OCIA
The protein set forth in SEQ ID NO: 14: AAH01837 (GBAS)
glioblastoma amplified sequence; GBAS
Protein described in SEQ ID NO: 15: NP-149061 (cat eye c5-2)
cat eye syndrome chromosome region,
candidate 5 isoform 2 precursor

Group B: Proteins that have knowledge about some of the functions, but whose functions in mitochondria are not clear.
Protein described in SEQ ID NO: 16: NP-006308 (BASP1)
brain abundant, membrane attached signal protein 1
Protein described in SEQ ID NO: 17: NP-002083 (GRSF1)
G-rich RNA sequence binding factor 1
Group C: localization to mitochondria is clear, and there is knowledge about some of the functions,
Protein with no knowledge of its effect on mitochondrial morphology.
Protein described in SEQ ID NO: 18: NP-002625 (PHB)
prohibitin; PHB
The protein of SEQ ID NO: 19: NP-038470 (SLP2)
stomatin-like protein 2; SLP2
The protein described in SEQ ID NO: 20: NP-056195 (CGI-51)
CGI-51 protein
Protein described in SEQ ID NO: 21: NP-006109 (HAX-1)
HS1 binding protein; HAX1
Protein described in SEQ ID NO: 22: NP-004484 (HADH2)
hydroxyacyl-Coenzyme A dehydrogenase, type II; HADH2

Of these proteins, the 15 types of proteins listed in Group A have been known for their gene base sequences by human genome analysis, but their functions as well as where the proteins exist and how It is completely unknown whether the protein exists in any form, and the protein itself has not yet been obtained by humans and has been isolated and identified for the first time by the present invention. Therefore, these proteins are novel proteins that humans have acquired for the first time and have confirmed their actions.
Moreover, although the sequences of SEQ ID NOs: 13 to 15 in the group A have been estimated in genome studies and the like, they have not been isolated and confirmed so far, This protein has been estimated to be related to other diseases. However, these proteins themselves have not yet been obtained by humans, and have been isolated and identified for the first time by the present invention. Therefore, these proteins are novel proteins that humans have acquired for the first time and have confirmed their actions. For example, a protein named NP-060300 of SEQ ID NO: 13 is a gene that is conformed to chromosome 4p11 by immunoscreening from an ovarian cancer cDNA library (Biochem. Biophys. Res. Commun. 280 (1), 401-406 (2001)). The protein named AAH01837 of SEQ ID NO: 14 was estimated from analysis of cDNA having a total length of 15000 or more in humans and mice (Proc. Natl. Acad. Sci. USA 99 (26), 16899- 16903 (2002)). Furthermore, a protein named NP-149061 of SEQ ID NO: 15 is a candidate for the critical region of human cat eye syndrome and the conserved synteny of mice and the centromere around chromosome 22 This is estimated from gene search (Genome Res. 11 (6), 1053-1070 (2001)).

On the other hand, proteins classified into Group B and Group C have been isolated and confirmed, and their functions have been clarified to some extent. For example, a protein named NP-006308 described in SEQ ID NO: 16 of Group B is a BASP1 family, which is a protein esterified with myristic acid at the axon end, and analyzes of its primary structure and physicochemical properties. Studies have also been reported (Biochimie 79 (6), 373-384 (1997)), which is a membrane-bound protein with transient phosphorylation sites and PEST motifs. In addition, a protein named NP-002083 described in SEQ ID NO: 17 is reported as GRSF-1 which is a poly (A) + mRNA binding protein related to a conserved G-rich element (Nucleic Acids Res. 22 (12), 2334-2343 (1994)), this protein is localized in the cytoplasm, and some variations due to splicing have been reported, but the full length has not yet been reported. is there.
The proteins classified into the group C have already been reported to some extent, but it has not yet been reported that these functions are related to mitochondrial function.
Group C proteins have already been clarified in the mitochondria, and some knowledge about the function has been reported, but it has been reported that these proteins affect the mitochondrial morphology. There is no protein.

  The result of considering the domain structure from the amino acid sequences of these proteins is shown in FIG. 5 and FIG. FIG. 5 shows the domain structure of the proteins of SEQ ID NOs: 1 to 12, FIG. 6 shows the domain structure of the proteins of SEQ ID NOs: 13 to 23, and the names on the left side of each figure are the names of the proteins of the respective sequence numbers Is shown. That is, in each protein of the above-mentioned sequence number, it displays with the name shown in the parenthesis. The numbers on the right side of each box in FIGS. 5 and 6 indicate the number of amino acids in each protein. The lengths of the boxes in FIG. 5 and FIG. 6 show the outline of the length of the amino acid length, the hatched portion in each box indicates a weak mitochondrial target signal (weak MTS), and the hatched portion indicates the mitochondrial target Indicates a signal (MTS), black filled area (blue in the original figure) indicates a strong transmembrane domain (strong TM), gray filled area (original figure in light blue) indicates a weak transmembrane domain (weak TM) Indicates that “CC” in FIGS. 5 and 6 indicates a coiled-coiled domain, “mTERF” indicates a conserved sequence of mitochondrial transcription termination factor, and “ANK rep” “ATPase” indicates an ATP-degrading enzyme conserved sequence, “myr” indicates a myristylation site conserved sequence, and “PEST” indicates a protein degradation signal. “RRM” indicates an RNA recognition motif sequence, and “Bac.Surface Ag” indicates a conserved sequence of a bacterial surface antigen (Bacterial surface antigen), respectively. .

In order to further elucidate the functions of these proteins, a gene encoding a group A protein was isolated, a gene fused with a FLAG tag was subcloned into an eukaryotic expression vector, and then cultured. The localization of the gene product introduced into the cells and the effect on the mitochondrial morphology were examined by immunostaining.
For this purpose, genes encoding c6orf125 (SEQ ID NO: 7), E3-3a (SEQ ID NO: 9), and LOC79135 (SEQ ID NO: 2), which belong to the group A, are introduced into HeLa cells with FLAG tags, respectively. This was expressed, and immunostaining by double staining was examined using an antibody against cytochrome C (α-cyt.c) and a tag-labeled protein (α-FLAG). The results are shown in FIGS. The photograph on the left side of FIG. 7 is a micrograph of mitochondria in each cell. The second photograph from the left shows a dye (red) stained with an antibody against cytochrome C (α-cyt.c), and the third photograph from the left is stained with a tag-labeled protein (α-FLAG) (green). The photo on the right is a merge of these. The photograph of FIG. 8 shows the enlarged image of FIG.
As a result, c6orf125 (SEQ ID NO: 7), E3-3a (SEQ ID NO: 9), and LOC79135 (SEQ ID NO: 2) are all localized in mitochondria. In c6orf125 and E3-3a, the mitochondria are tubular. It is considered that the protein is involved in mitochondrial fusion and division, and in LOC79135, the mitochondrion becomes an axle-like form. This protein was shown to be active in inducing mitochondria into an axle-like form.

Further, among the proteins belonging to Group A, LOC79714 (SEQ ID NO: 5), CHCHD3 (SEQ ID NO: 10), and OCIA (SEQ ID NO: 13) were similarly examined by immunostaining. The result is shown in FIG. The photograph on the left side of FIG. 9 is a micrograph of mitochondria in each cell. The second photograph from the left shows a dye (red) stained with an antibody against cytochrome C (α-cyt.c), and the third photograph from the left is stained with a tag-labeled protein (α-FLAG) (green). The photo on the right is a merge of these.
As a result, all the proteins of LOC79714 (SEQ ID NO: 5), CHCHD3 (SEQ ID NO: 10), and OCIA (SEQ ID NO: 13) are localized in mitochondria. In LOC79714 and CHCHD3, mitochondria aggregate near the cell nucleus. These proteins have been shown to have the activity of aggregating mitochondria within cells.

  Next, each protein of PHB (SEQ ID NO: 18), SLP2 (SEQ ID NO: 19), HADH2 (SEQ ID NO: 22), CGI-51 (SEQ ID NO: 20), and HAX-1 (SEQ ID NO: 21) belonging to group C is obtained. The encoded gene was immunostained using the same FLAG tag. For immunostaining of PHB, SLP2, and HADH2, the protein OPA1 (OPTIC ATROPHY) localized in the mitochondrial intermembrane space (between the inner and outer membranes) is used instead of cytochrome C antibody (cyt. C) as an antibody. The antibody (α-OPA1) against 1) was used. The result is shown in FIG. The upper three rows of FIG. 10 show the results of PHB, SLP2, and HADH2, respectively, which are stained green with OPA1 antibody (α-OPA1) and stained red with α-FLAG tag protein. The lower two rows of FIG. 10 show the results of CGI-51 and HAX-1, which are stained in red with an antibody against cytochrome C (α-cyt.c), and with tag-labeled protein (α-FLAG). It is dyed green. Moreover, the left photograph of FIG. 10 is a micrograph of mitochondria in each cell. The second photograph from the left is stained with OPA1 antibody (α-OPA1) (green) (upper three rows) or stained with antibody against cytochrome C (α-cyt.c) (red) (lower 2). The third photograph from the left shows the one stained with tag-tagged protein (α-FLAG) (red) (upper three), or the one stained with tag-tagged protein (α-FLAG) (green) (Bottom two steps) is shown, and the photograph on the right is a merged version. As a result, it was shown that any of the proteins belonging to Group C of the present invention has an activity to aggregate mitochondrial morphology.

Next, changes in mitochondrial membrane potential were examined for PHB (SEQ ID NO: 18) and SLP2 (SEQ ID NO: 19) proteins belonging to Group C. A gene encoding a fusion protein in which a FLAG tag is fused to each carboxyl terminus of PHB and SLP2 is forcibly expressed in HeLa cells, and the mitochondrial membrane potential in these cells is detected by a mitochondrial staining marker Rh123 depending on the mitochondrial membrane potential did. The result is shown in FIG. The upper part of FIG. 11 shows the case of PHB, the middle part shows the case of SLP2, and the lower part-shows the case of normal control cells without gene transfer. The photograph on the left side of FIG. 11 shows the case of staining with the mitochondrial staining marker Rh123 (green), and the photograph on the right shows a micrograph.
As a result, although a decrease in mitochondrial membrane potential was not observed in cells overexpressing SLP2, a decrease in mitochondrial membrane potential was observed in cells overexpressing PHB (in the upper left photograph in FIG. 11). See arrow). This indicates that the PHB protein may be involved in the regulation of membrane potential in mitochondria, and some proteins in the present invention are related not only to the control of mitochondrial morphology but also to the control of mitochondrial membrane potential. It was shown that the protein is included.

As described above, the 22 proteins shown in the present invention are related to the control of mitochondrial morphology, and have the effect of forming mitochondrial membrane tissue and / or mitochondrial morphology or mitochondrial membrane morphology. It has a regulating / controlling action and is useful as a reagent for repairing and normalizing abnormalities in mitochondrial morphology, mitochondrial membrane morphology, and mitochondrial membrane structure.
Therefore, the present invention provides a novel protein belonging to the aforementioned group A. These 15 proteins of the present invention each have the amino acid sequence set forth in SEQ ID NOs: 1 to 15 in the sequence listing, and the amino acid sequence has an activity capable of controlling or regulating the mitochondrial morphology. In the total amino acid sequence described in the sequence listing, 50% or less of amino acids are substituted with other amino acids, and also 100% or less of amino acids are added, and / or Similarly, it may have an amino acid sequence in which 50% or less of amino acids are deleted. The protein of the present invention includes the proteins described in SEQ ID NOs: 1 to 15 in the sequence listing, and the amino acid sequence of the protein and 50% or more, preferably 70% or more, or 80% or more, more preferably 90% or more. And a protein having an activity capable of controlling or regulating mitochondrial morphology.
The present invention also provides a mitochondrial membrane tissue-forming agent composed of one or more proteins selected from the group of proteins belonging to the groups A to C described above, and a mitochondrial membrane morphology regulator.
Furthermore, the present invention is a cell in which one or more proteins selected from the group of 22 proteins belonging to the groups A to C described above are deficient or overexpressed, or a cell to which these proteins are added. A substance active to normalize the mitochondrial morphology, comprising adding a test substance to a cell having an abnormality in the mitochondrial morphology and measuring a change in the mitochondrial morphology by the test substance. A method for screening is provided.

Mitochondria are not only involved in the production of energy in the body, but also have a strong relationship with the supply of active oxygen and apoptosis, and various diseases such as dilated cardiomyopathy, myeloma, gastric cancer, and diabetes In addition, it is known to cause morphological changes due to nutritional disorders and aging. Twenty-two proteins of the present invention are strongly related to such changes in mitochondrial morphology, and these proteins can also control and regulate mitochondrial morphology.
Therefore, the mitochondrial membrane tissue-forming agent and the mitochondrial membrane morphology regulator of the present invention are effective for repairing various diseases caused by mitochondrial morphological abnormalities and abnormal mitochondrial morphologies caused by nutritional disorders and aging. In addition to being useful as a component of pharmaceutical compositions for treating and preventing various diseases caused by mitochondrial morphological abnormalities, it is also effective for preventing and recovering physical strength decline due to nutritional disorders and aging Useful as an ingredient. The pharmaceutical composition of the present invention comprises one or more proteins selected from the group of proteins belonging to the groups A to C of the present invention and a pharmaceutically acceptable carrier. The protein as the active ingredient in the pharmaceutical composition of the present invention may be the protein itself, but if necessary, an antibody against the protein, a gene encoding the protein, an antisense for suppressing the expression of the cranial protein, etc. There may be.

Examples of the “pharmaceutically acceptable carrier” in the pharmaceutical composition of the present invention include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners. , Thickeners, flavoring agents, solubilizers or other additives. The pharmaceutical composition of the present invention is formulated into tablets, powders, granules, injections, solutions, capsules, troches and the like by using one or more of the pharmaceutically acceptable carriers as necessary. can do. The pharmaceutical composition of the present invention is administered orally or parenterally, preferably parenterally. Examples of the dosage form for parenteral administration include injections and mucosal administration agents.
The dosage of the pharmaceutical composition of the present invention is the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, or active ingredients contained in the pharmaceutical composition (such as the above-mentioned proteins and antibodies thereof). The dose is usually in the range of 1 μg to 100 mg as an active ingredient per adult.

As the screening method of the present invention, the following (1) to (4),
(1) culturing cells in which one or two or more proteins selected from the group of 22 proteins belonging to the groups A to C described above are deficient or overexpressed, or cells to which these proteins are added,
(2) causing an abnormality in the form of mitochondria in the cultured cells;
(3) Add a test substance to the cells with abnormal mitochondrial morphology,
(4) Observe and measure changes in mitochondrial morphology in the cells by the test substance,
It consists of things.
As cells in this method, human cells, monkey cells, mouse cells, animal cells such as rat cells, microbial cells such as Escherichia coli and yeast can be used. Examples of a method for overexpressing the target protein in these cells include a method of transformation using a gene having one or two or more copy numbers. As a transformation method, a general method well known to those skilled in the art can be employed. In addition, the function of the target protein can be impaired using an antibody, or the expression of the target protein can be suppressed by deleting the gene. As a deletion method, a mutation method, a targeting method, or the like can be employed. Furthermore, depending on the cell type, the same kind of situation as the overexpression system of the target protein may be formed by adding the target protein to the culture system.
As a method for observing and measuring changes in mitochondrial morphology, various methods such as a method of observing with a microscope, an immunostaining method with an antibody of a target protein, a method with a tag protein, and a fluorescence method such as GFP can be adopted. it can.
Substances determined to have activity by the method for screening active substances for normalizing the mitochondrial morphology of the present invention are useful substances as active ingredients for various mitochondrial dysfunctions. Substances whose effectiveness has been confirmed are useful as active ingredients of pharmaceuticals and as ingredients in cosmetics and functional foods.

Antibodies against each of the above-described proteins of the present invention can also be produced. Therefore, the present invention provides antibodies, preferably specific antibodies, against the 22 proteins of the present invention described above.
These antibodies of the present invention can be produced by a known general production method. For example, the protein of the present invention or a protein comprising a part of the amino acid sequence thereof is used as an immunogen (antigen), optionally together with Freund's Adjuvant, a mammal, preferably a mouse, rat, hamster, guinea pig, It can be produced by immunizing a rabbit, chicken, cat, dog, pig, goat, horse or cow, more preferably a mouse, rat, hamster, guinea pig or rabbit. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. Monoclonal antibodies are obtained from antibody-producing cells (spleens, lymph nodes, bone marrow, tonsils, etc., preferably B cells of the spleen, preferably) and myeloma cells (myeloma cells) that are not capable of producing autoantibodies. Manufactured by preparing a hybridoma, cloning the hybridoma, and selecting a clone that produces a monoclonal antibody exhibiting specific affinity for the antigen used to immunize the mammal by immunoassay (such as ELISA) can do.

  The present invention provides a method for comprehensively identifying proteins that control or regulate mitochondrial function and morphology. For the first time, a protein having a function to control or regulate mitochondrial morphology is isolated by the method of the present invention. It became possible to specify. Therefore, the present invention provides a novel protein having a function of controlling or regulating mitochondrial morphology, and the mitochondrial membrane tissue-forming agent and mitochondrial membrane morphology for controlling or preparing the mitochondrial morphology according to the present invention A regulator is provided. The mitochondrial membrane tissue-forming agent and mitochondrial membrane shape regulator of the present invention normalizes the mitochondrial morphology, thereby normalizing mitochondrial function and enabling treatment and prevention of various diseases caused by mitochondrial abnormalities In addition, it is possible to recover the loss of mitochondrial function associated with nutritional disorders and aging, and to maintain the function.

FIG. 1 is a photograph replacing a drawing, showing two-dimensional electrophoresis of a mitochondrial matrix fraction obtained by the method of the present invention. FIG. 2 is a photograph replacing a drawing, showing two-dimensional electrophoresis of the matrix fraction obtained by the method of the present invention and the separated membrane fraction. FIG. 3 is a photograph instead of a drawing showing two-dimensional electrophoresis of proteins that loosely bind to the membrane obtained by washing the membrane fraction obtained by the method of the present invention with an alkaline solution. FIG. 4 is a photograph replacing a drawing showing two-dimensional electrophoresis of proteins having a property of firmly binding to a membrane such as having a transmembrane domain obtained by the method of the present invention. FIG. 5 schematically shows the number of amino acids of the proteins represented by SEQ ID NOs: 1 to 12 and their domain structures. FIG. 6 schematically shows the number of amino acids of the proteins represented by SEQ ID NOs: 13 to 23 of the present invention and their domain structures. FIG. 7 shows the introduction of genes containing genes encoding c6orf125 (SEQ ID NO: 7), E3-3a (SEQ ID NO: 9), and LOC79135 (SEQ ID NO: 2) of the present invention into HeLa cells. It is a color photograph which replaces drawing which shows the result of the immuno-staining when it was made to express. FIG. 8 shows the introduction of genes containing genes encoding c6orf125 (SEQ ID NO: 7), E3-3a (SEQ ID NO: 9), and LOC79135 (SEQ ID NO: 2) of the present invention into HeLa cells. It is a color photograph which replaces drawing which shows the result of the immuno-staining when it was made to express. FIG. 9 shows that genes containing genes encoding LOC79714 (SEQ ID NO: 5), CHCHD3 (SEQ ID NO: 10), and OCIA (SEQ ID NO: 13) among the proteins belonging to Group A of the present invention are respectively transferred to HeLa cells. It is a color photograph which replaces drawing which shows the result of the immuno-staining when it introduce | transduces and expresses. FIG. 10 shows PHB (SEQ ID NO: 18), SLP2 (SEQ ID NO: 19), HADH2 (SEQ ID NO: 22), CGI-51 (SEQ ID NO: 20), and HAX-1 (SEQ ID NO: 21) belonging to Group C of the present invention. 2 is a color photograph instead of a drawing showing the results of immunostaining when a gene containing a gene encoding each of the above proteins is introduced into HeLa cells and expressed. FIG. 11 shows the expression of genes containing the genes encoding PHB (SEQ ID NO: 18) and SLP2 (SEQ ID NO: 19) proteins belonging to Group C of the present invention when introduced into HeLa cells. 4 is a color photograph replacing a drawing, showing the result of detection of the mitochondrial membrane potential in the cells with the mitochondrial staining marker Rh123 depending on the mitochondrial membrane potential.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Isolation and assay of mitochondrial membrane fraction 5 × 10 ⁸ to 1 × 10 ⁹ HeLa cells cultured using a culture solution containing 10% FBS in DMEM were collected after trypsin treatment, and PCV (packed cell volume) was measured. . The cell mass was suspended in a 10 × PCV amount of hypotonic solution, homogenized with a potter type homogenizer, and then made isotonic with a sucrose solution. This was centrifuged at 600 g for 10 minutes at 4 ° C. in a high-speed cooling centrifuge, and the supernatant was used as the cytoplasm fraction. The obtained cytoplasm fraction was further centrifuged at 10,000 g for 10 minutes at 4 ° C. in a high-speed cooling centrifuge, and the precipitate was used as a crude mitochondrial fraction. This precipitate was suspended in an isotonic sucrose solution and then used for density gradient centrifugation. Density gradient centrifugation was performed using OptiPrep ^™ (Invitrogen) with a step gradient of 35% and 13% and 6% Percol (Difco) at 20000 rpm for 16 hours. Mitochondrial bands were obtained after centrifugation at 4 ° C. This was washed with an isotonic sucrose solution to obtain a pure mitochondrial fraction.
The obtained pure mitochondrial fraction was crushed with ultrasonic waves, centrifuged at 15000 rpm in a refrigerated microcentrifuge for 10 minutes to remove non-crushed mitochondria as a precipitate, and the supernatant was then removed at 100,000 rpm in a tabletop ultracentrifuge. The mixture was centrifuged at 4 ° C. for 20 minutes, the precipitate was used as a crude mitochondrial membrane fraction (A), and the supernatant was used as a matrix fraction (B). After the crude membrane fraction is treated with an alkaline solution, ultracentrifugation is performed under these conditions, and the precipitate is made into a protein group (C) that binds tightly to the mitochondrial membrane, and the supernatant is weakly bound to the mitochondrial membrane. Group (D).
In order to examine the possibility of fractionation of these mitochondrial fractions, two-dimensional electrophoresis was examined at each purification stage. Two-dimensional electrophoresis was performed by the following method. First, about 200 mg of protein in each fraction was purified by TCA precipitation, and solubilized with a sample buffer containing 7M urea and 2M thiourea. This was subjected to one-dimensional electrophoresis using a non-linear isoelectric focusing gel (Immobiline ™ DryStrip pH3-10 NL: Amersham Biosciences) in the pH 3-10 range, and then alkylated with iodoacetamide. It was subjected to a second-order electrophoresis. In the second-dimensional electrophoresis, 10% or 12% SDS-PAGE was performed, and protein spots were detected by silver staining or CBB staining.

FIG. 1 is a diagram developed using the above-described two-dimensional electrophoresis method using a crude mitochondrial membrane fraction (A) and FIG. 2 using a matrix fraction (B). In the second dimension, 10% SDS-PAGE was performed, and protein spots were detected by silver staining. As a result, when the two samples of FIGS. 1 and 2 are compared, the matrix fraction contains many soluble proteins (for example, HSP60: see arrows in FIGS. 1 and 2). It was found that the separation by the law was performed well.
FIG. 3 shows the results of examining the protein group (C) that binds strongly to the mitochondrial membrane by the same method, and FIG. 4 shows the results of examination of the protein group (D) that similarly binds weakly to the mitochondrial membrane. The gel was stained by CBB staining. As a result, membrane proteins (for example, ATP synthase d subunit: see arrows in FIGS. 3 and 4) are often found in the protein group (C) that binds strongly to the membrane. I found that it was done well.

Two-dimensional development of protein groups that strongly bind to the mitochondrial membrane and comprehensive identification of proteins In order to comprehensively analyze the protein group (C) that strongly binds to the mitochondrial membrane obtained in Example 1 above, The fractions were subjected to two-dimensional electrophoresis using 12% SDS-PAGE, and the protein was detected by enhancing the staining property of CBB staining. As a result, a total of 247 spots were detected (see FIG. 3).
All of these spots were excised and digested in gel with trypsin, and then the peptide was purified, and this was purified by LC-MS / MS (Paradigm MS-4 and MS / MS as nano LC). And Finigan LTQ was used in combination, and a database search was performed using the peptide sequence tagging method to identify all proteins in each spot. 247 spots were analyzed and 171 proteins were identified by eliminating duplicates. The results are classified in Table 1. From the above results, it was shown that the identified protein is a mitochondrial protein, and the purity of the purified mitochondrial preparation described in Example 1 is high.
Among the proteins identified in Table 1, the following (A) to (A) to (A) to (C) are focused on the proteins that may be newly involved in the regulation of mitochondrial morphology and function. C) was classified into three groups.
A. A group of proteins whose functions have not been clarified.
B. A group of proteins that have knowledge about some of the functions, but whose functions in mitochondria are not clear.
C. A group of proteins with clear mitochondrial localization and knowledge of some of the functions but no knowledge of mitochondrial morphology.

Among these proteins, 15 types belonging to Group A (A-1 to A-15), 2 types belonging to Group B (B-1 to B-2), and 5 types belonging to Group C (C-1 to The results of searching for a protein having an amino acid sequence that matches the identified results for a total of 22 proteins of C-5) are shown below. The amino acid sequences based on the search results of each protein in these protein groups and the matched partial amino acid sequences (the portions underlined in the following amino acid sequences) are shown below. In the following display, each amino acid is indicated by a single letter of the amino acid.
(1) Protein A-1 (protein described in SEQ ID NO: 1):
Search result: NP_060336 hypothetical protein LOC54968
AA 'amino acid sequence match rate (protein coverage by AA's): 10.00%
Searched amino acid sequence and its matching part mlflargspw avelplcgrr talcaaaar
1
gprasvsras sssgsgpsgpva gwstgpsgaa
31
rllrrrpgraq ipvywegyvr flntpsdkse
61
dgrliytgnm ar avfgvk cf systriglt
91
flpyiftnn aisesvplpi qifigigmgs
121
ftvitpvllh fitkgyvir l yheattdtyk
151
aitnamlamae tstvfhqndv kipdak hvft
181
tfyak tksll vnpvlfpnre dyhlmgydk
211
eefillymeet sekhrkhddk
241 260
(The underlined portion indicates a sequence matching portion. The same applies hereinafter.)

(2) Protein A-2 (protein described in SEQ ID NO: 2):
Search result: NP_077027 hypothetical protein LOC79135
AA 'protein coverage by AA's: 13.13%
Searched amino acid sequence and its matched part mfkviqrsvg
1
pknsvk vdel slysvpegqs k yveear sql
31
eesisqlr hy cepittwcqe tysqtkpkkmq
61
slvqwgldsy dylqnapppgf fprlgvigfa
91
grigllarg skikklvyp gfmglaasly
121
ypqqaivfaq vsgerlydwg lrgivivedl
151
wkenfqkpgn vknspgtk
181 198

(3) Protein A-3 (protein described in SEQ ID NO: 3):
Search result: NP_001001692 FLJ45139 protein
AA 'amino acid sequence match rate (protein coverage by AA's): 4.41%
Searched amino acid sequence and its matching part mkkrfynakt vsillvkqqn nwaissqlhh
1
qlpmnlesiw lrmekeslpr slnfhghyy
31
mnwqtkfmml lk tvsipr sq aiftqpgls
61
gtpqgykqsh sargwaatls cwgkdgsrql
91
swvtagssfh ksdlis
121 136

(4) Protein A-4 (protein described in SEQ ID NO: 4):
Search results: NP_775932 hypothetical protein LOC285492
AA 'amino acid sequence match rate (protein coverage by AA's): 10.86%
Searched amino acid sequence and its matching part mrlpgapalp dadflvhlhf
1
flvripwasa lpdapalvi lektfpehat
31
crgcwvsgyl cwtpgnic sanggflkie
61
ntyrqhhhh mhrhthtd ksithctdt
91
htqsqrhr lt sallrlfiln aitdtsk fga
121
avffyvyclf adsvykffpf lppfr
151 175

(5) Protein A-5 (protein described in SEQ ID NO: 5):
Search results: NP_078937 hypothetical protein LOC79714
AA 'amino acid sequence identity (protein coverage by AA's): 13.16%
Searched amino acid sequence and its matching part mtrtlcspgp
1
algr alghsi qqr atstakt wdryeyefvg
31
lnvreaaqgk vteaekvfmv arglvreare
61
dlevhqaklk evrdrldrvs redsqylela
91
trehrmlqee krrltaylra edserek fsl
121
fsaavr eshe kertraertk nwsligsvlg
151
alignvagsty vnrvrlqelk allleaqk gp
181
vslqair eq assysrqqrd llnlmvdlr g
211
lvhaagpgqd sgsqagsppt r drdvdvlsa
241
alkeqshshr qvhscleglr eqldglektc
271
sqmagvvqlv ksaapgglve padgampsfl
301
leqgsmilal sdteqrleaq vnrntystyl
331
vtcvtfvatl pvlymlfkas
361 380

(6) Protein A-6 (protein described in SEQ ID NO: 6):
Search results: AAQ89151 HSAL5836
AA 'amino acid sequence match rate (protein coverage by AA's): 31.46%
Searched amino acid sequence and its matching part
mhsaallll lllllllrr ffdgsalr eg
1
gsrekpgpsr rrwaghsep wrsptlrsgp
31
gfpsyplgvp afvfispgps psqwalpcl
61 89

(7) Protein A-7 (protein described in SEQ ID NO: 7):
Search result: NP_115716 chromosome 6 open reading frame 125
AA 'protein coverage by AA's: 53.13%
The searched amino acid sequence and its matched part maasryrr fl klceewpvde tkrgrdlgay
1
lr qr vaqafr agentqvaep eacdqmyesl
31
ar lhsnyykh kypr prdtsf sglsleeyk l
61
ilstdtleel keidkgmwkk lqekfapkgp
91
edhka
121 126

(8) Protein A-8 (protein described in SEQ ID NO: 8):
Search results: BAC04486 unnamed protein product
AA 'amino acid sequence identity (protein coverage by AA's): 18.60%
Searched amino acid sequence and its matching part mksk lpppsg r lcshswrtg sgaapggsag
1
aaegffagtv qqardktar qrpmargsse
31
pespaarrfs ipgsvqghld avgk srsgdi
61
gsslrveagd k rtqasperq phcgahdaqg
91
ehheaqig
121 129

(9) Protein A-9 (protein described in SEQ ID NO: 9):
Search results: NP_951032 nuclear protein E3-3 isoform a
AA 'protein coverage by AA's: 35.33%
Searched amino acid sequence and its matching part materialalrsl yrarpslrcp pvelpwapr
1
ghr lspadde lyqrtrisll qreaaqamyi
31
dsyncsrgfmi ngnr vlgpca llphsvvqwn
61
vgshqlimited sfslwlwl rieivvvgtg
91
drterlqsqv lqamr qrgia vevqdtpnac
121
atfnflcheg rvtgaalipp pggtsltslg
151
qaq
181 184

(10) Protein A-10 (protein described in SEQ ID NO: 10):
Search result: NP_060282 coiled-coil-helix-coiled-coil-helix domain
containing 3; CHCHD3
AA 'protein coverage by AA's: 39.21%
Searched amino acid sequence and its matching part mggtttstr v tfedeneni tvvk gir lse
1
nvidr mkess psgsksqr ys gaygasvsde
31
elkrrvaeel alaqakk ese dqkrlkqake
61
ldrer aaane qltr ailer icseeeraka
91
khlarqleek drvlk kqdaf ykeqlar lee
121
rssefyr vtt eqyqkaaeev eak fkrish
151
pvcadlqaki lqcylenthq tlkcsalatq
181
ymhcvnhaqq smlekgg
211 227

(11) Protein A-11 (protein of SEQ ID NO: 11):
Search result: NP_057026 hypothetical protein LOC51001; CGI-12
AA 'amino acid sequence match rate (protein coverage by AA's): 41.25%
Searched amino acid sequence and its matching part malsaqqpr wfnsvklrsl inaaqltkrf
1
trpartllhg fsaqpqissd ncflqwgfkt
31
yrtsslwns qstssssqen nsaqssllps
61
mneqsqktqn issfdselfl edeldelpls
91
pmqpiseeea iqiiadpplp pastlrd yv
121
dhsetlqklv llgvdlskie khpeaannll
151
rldfekdik q mllflk dvgi ednqlgaflt
181
knhaifsedl enlktrvayl hsknfskadv
211
aqmvrkapfl lnfsver ldn rlgffqk ele
241
lsvkk trdlv vrlpr lltgs levvkennmk v
271
yr llgfkhn eiqhmitr ip kmltankmkl
301
tetdfvhnv msifhiivk fpqvfntr lf
331
kvkerhlft ylgr aqydpa kpnyisldk l
361
vsipifif eak asvqdf ek flktl
391 417

(12) Protein A-12 (protein described in SEQ ID NO: 12):
Search results: Q9H078 suppressor of potassium transport defect 3; SKD3
AA 'protein coverage by AA's: 30.41%
Searched amino acid sequence and its matching part MLGSLVLRRK ALAPRLLLLRLRSPTLRRGHG
1
GASGRNVTTG SLGEPQWLRV ATGGRPGTSP
31
ALFSGRGAT GGRQGGRFDT KCLAAATWGR
61
LPGPEETLPG QDSWNGVPSR AGLGMCALAA
91
ALVVHCYSK S PSNKDAALLLE AAR ANNMQEV
121
SR LLSEGADV NAK HRLGWTA LMVAAINRNNN
151
SVVQVLLAAG ADPNLGDDFS SVYKTAKEQG
181
IHSLEDGGQD GASRHITNQW TSALEFRRWL
211
GLPAGVLITR EDDFNNNRLNN RASFK GCTAL
241
HYAVLADDYR TVKELLDGGA NPLQRNEMGH
271
TPLDYAR EGE VMKLLR TSEA KYQEKQR KRE
301
AEER RRFPLE QRRLHEHIGQ ESAIATVGAA
331
IR RKENGWYD EEHPLVFLFL GSSGIGKTEL
361
AKQTAKYMHK DAKKGFIR LD MSEFQERHEV
391
AKFIGSPPGY VGHEEGGQLT KK LKQCNPAV
421
VLFDEVDKAH PDVLTIMMLQL FDEGRLTDGK
451
GKTIDCKDA FIMTSNVASD EIAQHALQLR
481
QEAEMSRNR IAENLGGDVQI SDKITSKNF
511
KENVIRPILK AHFRRDEFLG RINEIVYFLP
541
FCHSELIQLV NKELNFWAKR AKQRHNITLL
571
WDREVADVLV DGYNVHYGAR SIKHEVERR V
601
VNQLAAAYEQ DLLPGGCCTLR ITVEDSDKQL
631
LKSPELPSPQ AEK RLPK LRL EIDKDSKTR
661
RLDIRAPLHP EKVCNTI
691 707

(13) Protein A-13 (protein described in SEQ ID NO: 13):
Search results: NP_060300 ovarian carcinoma immunoreactive antigen; OCIA
AA 'protein coverage by AA's: 13.47%
Searched amino acid sequence and its matching part mngradfrep naevprpiph
1
errvfaecnd esfwfrsvpl aatsmlitqg
31
liskissil pkygsipkli lacymyfag
61
klsyvktcqe kfk klenspl gealr sgqar
91
rssppghyyq k skydssvsg qssffvtspaa
121
dniemlphye pipfsssmne saptgitdhi
151
vqgpdpnlee spkrknity errnknr esy
181
evsltqk tdp svrpmhervp kkevkvnkyg
211
dtwde
241 245

(14) Protein A-14 (protein described in SEQ ID NO: 14):
Search results: AAH01837 glioblastoma amplified sequence; GBAS
AA 'amino acid sequence match rate (protein coverage by AA's): 9.44%
Searched amino acid sequence and its matching part maarvllag awagglllqr aapcsllprl
1
rtwtsssnrs redswlkslf vrkvdprkda
31
hsnlakket snlyklqfhn vkpecleayn
61
k icqevlpk i hedkhypctl vgtwntwyge
91
qdqavhlwry eggypaltev mnklr enkef
121
lefrrk arsdm llsrknqlll efsfwnepvp
151
r sgpniyerr syqlrpgtmi ewgnywarai
181
rfrqdgneav ggffsqigql ymvhhlwayr
211
dlqtredirn awhkhgwee lvytvpliq
241
emesrimipl ktsplq
271 286

(15) Protein A-15 (protein described in SEQ ID NO: 15):
Search results: NP_149061 cat eye syndrome chromosome region,
candidate 5 isoform 2 precursor
AA 'protein coverage by AA's: 3.87%
Searched amino acid sequence and its matching part maawgcvaal gaarglcwra araaaglqgr
1
parrcyavgp aqspptfgfl ldidgvlvrg
31
hr vipaalk a frrlvnsqgq lrvpvvfvtn
61
agnilqhska kelsallgce vdadqvilsh
91
spmklfseyh ekrmlvsgqg pvmenaqglg
121
fr nvvtvdel r mafpllldmv derrrlkttp
151
lprndfpri gvlllgepvr wetslqlimd
181
vllsngspga glatppyph pvlasnmdl
211
wmaeakmprf ghgtflcle tieqkvtgke
241
lryeglmmgkp siltyqyaed lirrqaerg
271
waapirklya vgdnpmsdvy ganlfhqylq
301
kathdgapel gagtrqqqp sasqscisil
331
vctgvynprn pqstepvlgg geppfhghrd
361
lcfspglmea shvvndvnea vqlvfrkegw
391
ale
421 423

(16) Protein B-1 (protein described in SEQ ID NO: 16):
Search results: NP_006308 brain abundant, membrane attached
signal protein 1
AA 'protein coverage by AA's: 76.21%
Searched amino acid sequence and corresponding part mggklskkk
1
teeegtpkes epqaaaepae akegk ekpdq
31
daegkaeeeke gekdaaake eapkaepekt
61
egaeaak aep pkapeqeqa pgpaaggeap
91
kaeaaaaapa esapaagee pskeegepkk
121
teapaapaq etksdgapas dskpgssea
151
pssketpaat eapstpkaq gpaasaeeepk
181
pveapaandd qtvtvke
211 227

(17) Protein B-2 (protein described in SEQ ID NO: 17):
Search result: NP_002083 G-rich RNA sequence binding factor 1
AA 'amino acid sequence identity (protein coverage by AA's): 13.92%
Searched amino acid sequence and its matching part magtrwvlga llrgcgcncs scrrtgaacl
1
pffyasaypa lrasllpqsl aaaavptrs
31
ysquestty edlppppeye lapskleeev
61
ddvfliraqg lpwssctmedv lnffsdcrir
91
ngengihfl nr dgkr rgda liemeseqdv
121
qk alekhrmy mgqryvevey innervdalm
151
kslqvk sspv vndgvvr lrg lpyscnekdi
181
vdffgllniv dtfvmdyrg rrktgeayvq
211
FEPEMANQUA llkhreeign r yieifpsrr
241
nevrthvsy kgkkiasfpt akiitepemv
271
FEHEVENNEY qpmtafesk eilpkevpe
301
klpeadfgt tsslhfvhmr glpfqanaqd
331
iinffaplkp vritmeiss gkatgeadvh
361
hetedavaa mlkdrshvhh r yielflnsc
391
pk gk
421 424

(18) Protein C-1 (protein described in SEQ ID NO: 18):
Search result: NP_002625 prohibitin; PHB
AA 'protein coverage by AA's: 29.04%
The searched amino acid sequence and its matching part maakvfesig kfglalavag gvvnsalynv
1
daghr avifd rfr gvqdivv gegthflipw
31
vqkpiifdcr srpr nvpvit gskdlqnvni
61
tlr ilfrpva sqlpriftsi gedydrvlp
91
siteilksv var fdageli tqr elvsrqv
121
sdlterata fglildvsl thltfgk eft
151
eaveakqvaq qeaarfvv ekaeqqkkaa
181
iisaegdsk a aileanslat aggdglielrk
211
leaeadiaq lsrsrnityl pagqsvllql
241
pq
271272

(19) Protein C-2 (protein described in SEQ ID NO: 19):
Search result: NP_038470 stomatin-like protein 2; SLP2
AA 'amino acid sequence match rate (protein coverage by AA's): 41.01%
Searched amino acid sequence and its matching part mlaraargg
1
glpr ntvvlf vpqqeawvve r mgrfhill
31
pglniripvl dryvqslk evinvpeqs
61
avtdnvtlq idgvlyrim dpyka sigve
91
dpeyavtqla qttmrsselgk lsldkvfr er
121
eslnasivda inqadcwgi r cryyeikdi
151
hvpprvkesm qmqveer rk ratvlegegt
181
resinvaeg kkqaqilase aekainqa
211
ageasaavlak aka akaeairi laaaltqhng
241
daasltvae qyvsafskla kdsntilps
271
npgdvtsmva qmgvygalt k apvpgptds
301
lssgsssr dvq gtdasldeel drvkms
331 356

(20) Protein C-3 (protein described in SEQ ID NO: 20):
Search result: NP_056195 CGI-51 protein
AA 'amino acid sequence identity (protein coverage by AA's): 9.38%
Searched amino acid sequence and its matching part mgtvharsl plpssgpdfg
1
veepakqeil enkdvvvqhv hfdglgrtkd
31
dicieigdv fkkanlievm rkshearekl
61
lr lgifr qvd vlidtcqgdd alpngldvtf
91
evtelrrrlg syntmvgne gsmvlglklp
121
nllgraekvt fqfsygtket sglsffkpr
151
pgnfer nfsv nlykvtgqfp wsslr etdrg
181
msayysfpiw ktshtvkweg vwrergclsr
211
tasfavrkes ghslksslsh amvidsr nss
241
ilpr rgallck vnquelagtg gdvsfikedf
271
elqlnqqlif dsvfsasfwg gmlvpigdkp
301
ssiadrfylgptsvrgfsm hsigqqsegd
331
ylggeaywag glhlytplpf rpgqggfgel
361
frthfflnag nlcnlnnygeg pkahirklae
391
cirwsygagi vlrggnial elnycvpmgv
421
qtgdr icdgv qfgagir fl
451 469

(21) Protein C-4 (protein described in SEQ ID NO: 21):
Search result: NP_006109 HS1 binding protein; HAX1
AA 'protein coverage by AA's: 18.64%
Searched amino acid sequence and its matching part mslfdllfrgf fgfppgprshr dffgggmtrd
1
edddeeeee ggswgrgnpr fhspqhppee
31
fgfgfsfspg ggilfhdnfg fddlvrdfns
61
ifsdmmgawtl pshppelpgp setpger lr
91
egqtlr dsml kypdshqpr i fggvlesdar
121
sespqpapdw gsqrpfhrfd dvwpmpdphpr
151
trendndldsq vsqeglgpvl qpqpksyfk s
181
isvkitkpd giberertrvv dsegrttv
211
tr headspr gdpesprpppa lddafsildl
241
flgrwfrsr
271 279

(22) Protein C-5 (protein described in SEQ ID NO: 22):
Search results: NP_004484 hydroxyacyl-Coenzyme A dehydrogenase,
type II; HADH2
AA 'protein coverage by AA's: 46.36%
Searched amino acid sequence and its matching part maaacrsvk g lvavitggas glglataerl
1
vgqgasavll dlpnsggeaq akk lgncvv
31
apadvsekd vqtalalakg kfgr vdvavn
61
cagiavask t ynlk kgqtht ledfqr vldv
91
nlmgtfnvir lvagemgqne pdqggqrgvi
121
intasvaaf gqvgqaaysa sk ggivgmtl
151
pear drapig ir vmtiapggl fgtplltslp
181
ekvccnflasq vpfpsrllgdp aeyahlvqai
211
ienpflngev irldgairmq p
241 261

Among these proteins, 15 types belonging to Group A (A-1 to A-15), 2 types belonging to Group B (B-1 to B-2), and 5 types belonging to Group C (C-1 to The amino acid sequences based on the search results for a total of 22 proteins of C-5) are shown in SEQ ID NOs: 1 to 22 in the sequence listing, and their base sequences are shown in SEQ ID NOs: 23 to 44.
The domain structure of these proteins was predicted by using an expert protein analysis system (ExPASy), which is a proteomics server of SIB (Swiss Institute of Bioinformatics) for each protein, and predicted using various prediction tools. The results are shown in FIGS. From these results, it was revealed that many of the identified proteins are proteins having a predicted mitochondrial target signal (MTS), a transmembrane domain, and the like.

Isolation of cDNA of mitochondrial membrane protein group and examination of effect on mitochondria by overexpression in cell Each cDNA of protein group isolated and identified in Examples 1 and 2 was isolated by PCR method, and A gene encoding a fusion protein that fuses a tag protein such as a FLAG tag to the carboxyl terminus was constructed, and a plasmid was prepared by inserting this into an expression vector for animal cells. These genes were introduced into HeLa cells and expressed. These expressed cells were double-stained by immunocytostaining using an antibody against cytochrome C (cyt. C) as a tag-labeled protein and a mitochondrial protein. This was observed with a confocal laser microscope.
The results are shown in FIG. An enlarged view of the result is shown in FIG. As a result, c6orf125 and E3-3a are located at the tip of a mitochondrion having a tubular form and may be involved in mitochondrial fusion and division. LOC79135 was shown to have an activity to induce mitochondria to take an axle-like form.
Moreover, the result of having similarly performed LOC79714, OCIA, and CHCHD3 as protein is shown in FIG. As a result, it was shown that OCIA and CHCHD3 proteins were localized in mitochondria, and that LOC79714 and CHCHD3 had activity to aggregate mitochondria.

The same immunostaining as in Example 3 was performed using PHB, SLP2, and HADH2, and CGI-51 and HAX-1 as proteins. As for PHB, SLP2, and HADH2, cyt. Instead of C, an antibody of OPA1 was used.
The result is shown in FIG. As a result, it was shown that each protein has an activity to aggregate mitochondrial morphology.

Effects on mitochondrial membrane potential PHLa which is a protein belonging to group C and a gene encoding a fusion protein in which a FLAG tag is fused to the carboxyl terminus of SLP2 are forcibly expressed in HeLa cells, and mitochondrial staining depending on mitochondrial membrane potential Mitochondrial membrane potential was detected with the marker Rh123.
The results are shown in FIG. As a result, although a decrease in mitochondrial membrane potential was not observed in cells overexpressing SLP2, a decrease in mitochondrial membrane potential was observed in cells overexpressing PHB (arrows in FIG. 11). This indicates that PHB protein may be involved in the regulation of membrane potential in mitochondria.

  The present invention provides a novel protein having a function capable of controlling or regulating mitochondrial morphology, a mitochondrial membrane tissue-forming agent and a mitochondrial membrane morphology regulator using the protein, and the mitochondrial morphology. It has industrial applicability not only as a pharmaceutical composition such as a therapeutic agent or preventive agent for various diseases caused by abnormalities in the body, but also as a material for cosmetics and functional foods to maintain the form and function of mitochondria. Is.

SEQ ID NO: 1: NP-060336 amino acid sequence.
SEQ ID NO: 2: amino acid sequence of NP-0707027.
SEQ ID NO: 3: amino acid sequence of NP-001001692.
SEQ ID NO: 4: amino acid sequence of NP-775932.
SEQ ID NO: 5: amino acid sequence of NP-078937.
SEQ ID NO: 6: AAQ89151 amino acid sequence.
SEQ ID NO: 7: amino acid sequence of NP-115716.
SEQ ID NO: 8: Amino acid sequence of BAC04486.
SEQ ID NO: 9 amino acid sequence of NP-951032.
SEQ ID NO: 10: amino acid sequence of NP-060282.
SEQ ID NO: 11 amino acid sequence of NP-057026.
SEQ ID NO: 12: amino acid sequence of Q9H078.
SEQ ID NO: 13 amino acid sequence of NP-060300.
SEQ ID NO: 14: Amino acid sequence of AAH01837
SEQ ID NO: 15: amino acid sequence of NP-149061.
SEQ ID NO: 16: amino acid sequence of NP-006308
SEQ ID NO: 17 amino acid sequence of NP-002083.
SEQ ID NO: 18: amino acid sequence of NP-002625.
SEQ ID NO: 19: amino acid sequence of NP-038470.
SEQ ID NO: 20: amino acid sequence of NP-056195.
SEQ ID NO: 21: amino acid sequence of NP-006109.
SEQ ID NO: 22: amino acid sequence of NP-004484.
Sequence number 23: The base sequence of NP-060336.
SEQ ID NO: 24: Nucleotide sequence of NP-0707027.
Sequence number 25: The base sequence of NP-001001692.
SEQ ID NO: 26: nucleotide sequence of NP-775932.
Sequence number 27: The base sequence of NP-078937.
SEQ ID NO: 28: AAQ89151 base sequence.
Sequence number 29: The base sequence of NP-115716.
SEQ ID NO: 30: Base sequence of BAC04486.
SEQ ID NO: 31: nucleotide sequence of NP-951032.
SEQ ID NO: 32: base sequence of NP-060282
SEQ ID NO: 33: Nucleotide sequence of NP-057026.
SEQ ID NO: 34: base sequence of Q9H078.
Sequence number 35: The base sequence of NP-060300.
SEQ ID NO: 36: base sequence of AAH01837 SEQ ID NO: 37: base sequence of NP-149061
SEQ ID NO: 38: base sequence of NP-006308
SEQ ID NO: 39: nucleotide sequence of NP-002083.
Sequence number 40: The base sequence of NP-002625.
SEQ ID NO: 41: nucleotide sequence of NP-038470.
SEQ ID NO: 42: nucleotide sequence of NP-056195.
Sequence number 43: The base sequence of NP-006109.
Sequence number 44: The base sequence of NP-004484.

Claims (3)

The amino acid sequence according to any one of SEQ ID NOs: 2, 7, and 9 in the sequence listing, or an amino acid sequence having at least 90% identity with these amino acid sequences, and the ability to form mitochondrial membrane tissue, An agent for controlling or regulating the form of mitochondria, which contains as an active ingredient a protein having an activity of inducing an abnormal tubular or axle-like form. It has an amino acid sequence described in any one of SEQ ID NOs: 2, 7, and 9 in the sequence listing, or an amino acid sequence having at least 90% identity with these amino acid sequences, and has the ability to form mitochondrial membrane tissue An agent for controlling or regulating the form of mitochondria comprising, as an active ingredient, a gene encoding a protein having an activity to induce mitochondria into an abnormal tubular or axle-like form. It has an amino acid sequence described in any one of SEQ ID NOs: 2, 7, and 9 in the sequence listing, or an amino acid sequence having at least 90% identity with these amino acid sequences, and has the ability to form mitochondrial membrane tissue A cell in which one or more proteins selected from the group of proteins having an activity to induce mitochondria into an abnormal tubular or axle-like form is deficient or overexpressed, or a cell to which these proteins are added. A substance active to normalize the mitochondrial morphology, comprising adding a test substance to a cell having an abnormality in the mitochondrial morphology and measuring a change in the mitochondrial morphology by the test substance. How to screen.