JP2007137821A - Rab27a deactivator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a control factor of Rab27A which engages in the transport of a melanosome. <P>SOLUTION: This Rab27A deactivator contains either one of the following (a)-(d): (a) a protein consisting of an amino acid sequence expressed with sequence number 2, or a protein consisting of an amino acid sequence deleted, permuted, or added with one or a few amino acids in a protein consisting of an amino acid sequence expressed with sequence number 2, and having Rab27A GTP-ase activity promoting action; (b) a protein consisting of an amino acid sequence expressed with sequence number 4, or a protein consisting of an amino acid sequence deleted, permuted, or added with one or a few amino acids in a protein consisting of an amino acid sequence expressed with sequence number 4, and having Rab27A GTP-ase activity promoting action; (c) a gene coding the (a) or (b) protein; and (d) a recombinant vector containing the gene coding the above (a) or (b) protein. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the use of a protein capable of inactivating Rab27A involved in melanosome transport and the like.

  The low molecular weight G protein Rab is universally conserved from yeast to human as a factor controlling membrane transport. There are more than 60 types of Rab in humans, each of which is thought to regulate its unique membrane traffic. Rab takes two forms, GTP-bound activated form (GTP-Rab) and GDP-bound deactivated form (GDP-Rab), and cycling of these two states is responsible for its function in membrane trafficking. Is essential. The activated GTP-Rab promotes membrane transport by binding to a specific binding protein (effector molecule). Rab27A is the first Rab to be associated with a human hereditary disorder, with hair whitening due to abnormal melanosome transport and severe immune deficiency due to abnormal release of lytic granules from cytotoxic T cells. It was identified as a causative gene product of type II Griscelli syndrome that presents symptoms (Non-patent Document 1). Therefore, Rab27A controls the transport of melanin pigment (melanosomes) in pigment cells (melanocytes) and plays an important role in darkening skin and hair, as well as being involved in various secretory phenomena including endocrine and exocrine secretion. It is believed that

  In order to convert and inactivate Rab from GTP type to GDP type, a factor called GTPase-activating protein (GAP) is required (Non-patent Document 2). Each Rab has a specific GAP (Rab-GAP), but only a few such as Rab5-GAP have been identified so far, and most Rabs including Rab27A have identified Rab-GAP. Not. Recently, it has been shown that a protein motif called TBC (Tre-2 / Bub2 / Cdc16) functions as Rab-GAP in yeast (Non-patent Document 3), and a gene having 50 or more TBC domains on the genome in humans. It became clear that there exists (nonpatent literature 4). However, many of these proteins with TBC domains can be difficult to purify recombinant proteins in large quantities, and it has been confirmed whether these proteins actually have Rab-GAP activity using conventional biochemical techniques. It was extremely difficult to do. Moreover, although it has been reported that Rab5-GAP was identified by a screening method for Rab-GAP using protein interaction utilizing the substrate specificity of Rab and Rab-GAP as an index (Non-patent Document 5). However, there is a limit in that there are many exceptions such as having no GAP activity even if binding is observed, or having GAP activity even if binding is not observed (Non-patent Document 6).

Menasche, G., E. Pastural, J. Feldmann, S. Certain, F. Ersoy, S. Dupuis, N. Wulffraat, D. Bianchi, A. Fischer, F. Le Deist, and G. de Saint Basile. 2000 Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nature Genet. 25: 173-176. Zerial, M., and H. McBride, 2001. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2: 107-117. Strom, M., P. Vollmer, T. J. Tan, and D. Gallwitz, 1993, A yeast GTPase-activating protein that interacts specifically with a member of the Ypt / Rab family.Nature 361: 736-739. Bernards, A., 2003. GAPs galore. A survey of putative Ras superfamily GTPase-activating proteins in man and Drosophila. Biochim. Biophys. Acta. 1603: 47-82. Haas, A. K., E. Fuchs, R. Kopajtich, and F. A. Barr., 2005. A GTPase-activating protein controls Rab5 function in endocytic trafficking. Nat. Cell Biol. 7: 887-893. Itoh, T., M. Satoh, E. Kanno, and M. Fukuda 2005.Screening for target Rabs of TBC (Tre-2 / Bub2 / Cdc16) domain-containing proteins based on their Rab-binding activity.Submitted for publication.

  As described above, for many Rabs including Rab27A, a specific Rab-GAP has not yet been identified.

  Therefore, an object of the present invention is to provide a regulator of Rab27A involved in melanosome transport and the like using a conventional biochemical method and a screening method using a new index different from protein interaction.

  As a result of intensive research to solve the above problems, the present inventors have focused on the fact that Rab27A is an essential factor for melanosome transport, and screened for proteins that inactivate Rab27A using melanosome transport abnormalities as indicators. As a result, the inventors succeeded in identifying two types of proteins that cause aggregation of melanosomes. Moreover, it was confirmed that these proteins actually have Rab27A-GAP activity at the cellular level and the test tube level. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
1. A Rab27A inactivating agent containing any one of the following (a) to (d):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Rab27A Protein with GTPase activity promoting action
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and Rab27A Protein with GTPase activity promoting action
(c) a gene encoding the protein of (a) or (b)
(d) Recombinant vector containing a gene encoding the protein (a) or (b) 2.1. A pharmaceutical agent for treating and / or preventing a disease associated with Rab27A activation, comprising the Rab27A inactivating agent described in 1 above as an active ingredient.
3. 1. A disease with Rab27A activation is pigmentation. The medicament according to 1.
4). A method for screening a substance that regulates the GTPase activity of Rab27A, comprising the following steps.
(1) A step of bringing GTP-bound Rab27A into contact with a test substance in the presence of any of the following proteins (a) or (b)
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Rab27A Protein with GTPase activity promoting action
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and Rab27A Protein with GTPase activity promoting action
(2) Process for monitoring RTP27A GTP hydrolysis
(3) A step of selecting a test substance that promotes or suppresses GTP hydrolysis as compared with a case where the test substance is not contacted. 3. Control of Rab27A GTPase activity is melanosome transport control, hormone secretion control, digestive enzyme secretion control, or granule release control from immune cells. The method described in 1.

  According to the present invention, a protein (Rab27A-GAP) having a GTPase activity promoting action of Rab27A, which is an essential factor for melanosome transport, has been identified. The Rab27A-GAP protein can inactivate Rab27A and suppress the transport of the melanosome from the periphery of the nucleus to the periphery of the cell membrane, and thus can be used as an active ingredient of a medicament for treating and / or preventing pigmentation, for example. It is also possible to screen for a substance that regulates the GTPase activity of Rab27A, using the degree of hydrolysis of GTP bound to Rab27A by the Rab27A-GAP protein as an index. A substance that regulates the GTPase activity of Rab27A obtained by such screening can promote or suppress melanosome transport involving Rab27A, secretion of hormones, secretion of digestive enzymes, granule release from immune cells, etc. It is useful as a medicament for treatment and / or prevention of various diseases such as skin whitening agents and white hair suppressants, as well as endocrine / exocrine systems and immune systems.

1. Rab27A Inactivating Agent The Rab27A inactivating agent of the present invention contains a substance having a GTPase activity promoting action of Rab27A.
As the above-mentioned substance having a GTPase activity promoting action of Rab27A, a protein that causes aggregation of melanosomes and in which Rab27A-GAP activity is recognized in vitro can be used. And a protein having the amino acid sequence represented by SEQ ID NO: 4 which is a homologue thereof (named “FLJ13130 / Rab27A-GAPβ”). .

  The above EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ proteins include mutant proteins that are functionally equivalent. Such a mutant protein is a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having an action of promoting GTPase activity of Rab27A, Or a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and a GTPase of Rab27A Examples include proteins having an activity promoting action.

  Here, the range of “one or several” which may be deleted, substituted or added is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, More preferably, it means 1 to 5, particularly preferably about 1 to 3.

  Deletion, addition and substitution of amino acids can be performed by modifying a gene encoding the protein using a technique known in the art. Mutation can be introduced into a gene by a known method such as Kunkel method, Gapped duplex method, Coupled primary cyclic selection method, Eckstein method, cassette mutation method or the like, for example, site-directed mutagenesis. Mutation can be performed using a mutagenesis kit (eg, Mutan-K (TAKARA) or Mutan-G (TAKARA)) or using TAKARA's LA PCR in vitro Mutagenesis series kit. be introduced.

  Here, the “promoting effect of Rab27A on GTPase activity” refers to the action of activating GTPase (ie, Rab27A itself), an enzyme that hydrolyzes GTP bound to Rab27A into GDP. “Having a promoting action” means that the action (activity) is substantially equivalent to the action (activity) of the protein having the amino acid sequence represented by SEQ ID NO: 2 or 4.

As a method for measuring this activity, any method can be used as long as it can measure the amount of phosphoric acid generated by the reaction GTP → GDP + phosphoric acid (Pi), but typically, a substance that develops color when bound to phosphoric acid. Is added to the reaction solution, and the absorbance is measured. P at the γ-position of GTP is labeled with a radioactive substance such as ³² P, and only the released ³² Pi is adsorbed to the filter to increase its radioactivity intensity. Examples include a method of measuring, or a method in which P at the α-position of GTP is labeled with ³² P, GTP and GDP are separated by thin layer chromatography, and the intensity of their radioactivity is measured.

  The mutant protein further includes a protein having homology with the amino acid sequence represented by SEQ ID NO: 2 or 4. The homologous protein means a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 2 or 4. It means the protein which has. To determine protein homology, algorithms described in the literature (Wilbur W.J., and Lipman D.J. 1983. Proc. Natl. Acad. Sci. USA 80: 726-730.) May be used.

  The above EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ proteins also include proteins (partial peptides) that are functionally equivalent to these proteins and have a partial sequence of the amino acid sequence of the proteins. Such a partial peptide may be a peptide containing a TBC (Tre-2 / Bub2 / Cdc16) domain part commonly found in proteins having Rab-GAP activity, and has the amino acid sequence represented by SEQ ID NO: 2. Examples include peptides containing at least the amino acid sequence up to positions 108 to 313 or the amino acid sequence up to positions 82 to 287 of the amino acid sequence represented by SEQ ID NO: 4.

  The above protein or a partial peptide thereof may be used in the form of a salt, preferably in the form of a physiologically acceptable acid addition salt, if necessary. Such salts include salts of inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, Citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The protein can be obtained by a method of chemically synthesizing based on a known amino acid sequence, an in vitro transcription from an expression vector, a method of isolation and purification as an expression product of a transformed cell by an expression vector, or the like.

  For example, when a protein is expressed by in vitro translation, an expression vector having an RNA polymerase promoter is added to an in vitro translation system such as a rabbit reticulocyte lysate or wheat germ extract containing an RNA polymerase corresponding to the promoter, Proteins can be produced in vitro. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II.

  When a protein is expressed in a microorganism such as Escherichia coli, a DNA fragment encoding the protein is assembled into an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism. Instead, an expression vector is prepared. By transforming a host cell with this expression vector, a transformant cell that expresses the protein can be obtained. By culturing this transformant, the target protein can be mass-produced from the culture. it can. Examples of the expression vector for E. coli include pET vector (Novagen), pTrxFUS vector (Invitrogen), pCYB vector (NEW ENGLAMD Bio Labs) and the like. Further, when a protein is expressed in a eukaryotic cell, a DNA fragment encoding the protein is inserted into a expression vector for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site, etc., and a recombinant vector Is made. If this vector is introduced into a eukaryotic cell, a transformed eukaryotic cell expressing the target protein can be obtained. As the expression vector, when the host cell is yeast, pESP-1 expression vector (STRATAGENE), pAUR123 vector (TAKARA), pPIC vector (Invitrogen), etc., and the host cell is an animal cell. In some cases, for example, pMAM-neo expression vector (manufactured by CLONTECH), pCDNA3.1 vector (manufactured by Invitrogen), pBK-CMV vector (manufactured by STRATAGENE) and the like can be exemplified. Eukaryotic cells include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, human fetal kidney cells (HEK293 cells), monkey kidney cells (COS7 cells), Chinese hamster ovary cells (CHO). Animal cells such as cells) can be used.

  In order to introduce the expression vector into cells, known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used. In order to isolate and purify the protein expressed in the transformed cell, known separation operations can be combined. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

  The partial peptide can be produced by a known peptide synthesis method or by cleaving the above protein with an appropriate peptidase (eg, trypsin, chymotrypsin, arginyl endopeptidase). As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used.

  As a component of the Rab27A inactivating agent of the present invention, a gene encoding the protein represented by SEQ ID NO: 2 or a gene encoding the protein represented by SEQ ID NO: 4 can also be used.

  Specific examples of the gene include a gene containing a DNA consisting of the base sequence represented by SEQ ID NO: 1 or a gene containing a DNA consisting of the base sequence represented by SEQ ID NO: 3. The above gene may be a functionally equivalent mutant gene. For example, it hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA represented by SEQ ID NO: 1 under a stringent condition. And a gene comprising a DNA encoding a protein having a GTPase activity promoting action of Rab27A or a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 3 under stringent conditions Examples thereof include a gene comprising a DNA that hybridizes and encodes a protein having a GTPase activity promoting action of Rab27A.

  Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a nucleic acid having high homology, that is, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more of homology with the nucleotide sequence represented by SEQ ID NO: 1 or 3. A condition is that the complementary strands of DNA consisting of a base sequence having a hybridize and the complementary strands of nucleic acids with lower homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

  The gene may be chromosomal DNA or cDNA. Chromosomal DNA can be obtained, for example, by preparing a library of chromosomal DNA from cells or the like and screening the library using a probe that hybridizes to the gene. In addition, cDNA can be obtained by a gene amplification technique such as RT-PCR using an RNA sample extracted from a cell (tissue) considered to express the above gene and using a primer that hybridizes to the above gene. .

  As the Rab27A inactivating agent of the present invention, a recombinant vector containing the above gene can also be used. Such a vector is effective when the Rab27A inactivating agent of the present invention is used for gene therapy. General genetic manipulations such as insertion of the gene into a vector can be performed according to a conventional method. Examples of vectors that can be used for gene therapy include adenovirus vectors and retrovirus vectors.

2. Medicament containing Rab27A inactivating agent as active ingredient The Rab27A inactivating agent has an action of promoting the conversion of active Rab27A (GTP-Rab27A) to inactive Rab27A (GDP-Rab27A). Therefore, the Rab27A inactivating agent can be used as a medicament for preventing and / or treating a disease associated with activation of Rab27A by mixing with other pharmaceutically acceptable carriers. Rab27A is involved in the mechanism by which melanosomes that store melanin pigments synthesized in melanocytes are transported from the periphery of the melanocytes to the cell membrane. Thereafter, the melanosomes transported to the cell membrane are transferred to adjacent keratinocytes (or hair matrix cells), and darken the skin (or hair). Therefore, a disease associated with activation of Rab27A typically includes pigmentation disease in which Rab27A is activated and melanosome transport is actively carried out. When the pharmaceutical of the present invention is used for the disease, Rab27A By controlling in the direction of inactivation, it is possible to prevent the occurrence of pigmentation such as melasma, blemishes and freckles. The cause of pigmentation may be any of ultraviolet rays, hormone balance, and poisoning.

  Rab27A is also present on secretory granules of various secretory cells, and is also involved in promoting secretion of hormones such as insulin, digestive enzymes such as amylase, immune granules containing histamine and the like. Examples of diseases of abnormal secretion accompanied by activation of Rab27A include allergy due to excessive histamine secretion from mast cells, and when the medicament of the present invention is used for the disease, by controlling Rab27A in the direction of inactivation, Can prevent and treat allergic diseases.

  The medicament of the present invention can be prepared and administered in various forms capable of incorporating Rab27A-GAP into cells, containing the Rab27A inactivating agent as a main component.

  In order to obtain a form capable of incorporating the Rab27A-GAP protein or gene into cells, there are the following means.

  In the case of protein preparations, by using a fusion protein in which a cell membrane-passing peptide is linked to the N-terminal side of Rab27A inactivating protein (Rab27A-GAP), the protein can be taken into the cell through the cell membrane. Can be a possible form. As the cell membrane-passing peptide, HIV-1 • TAT PTD (protein transduction domain) or Drosophila homeobox protein antennapedia PTD can be used. A fusion protein can be prepared by preparing a fusion DNA fragment obtained by linking a DNA fragment encoding a Rab27A inactivating protein and a cell membrane transit peptide, and expressing it in a host cell such as Escherichia coli. Alternatively, a fusion protein can be prepared via a divalent cross-linking agent (for example, EDC, β-alanine, etc.).

  In the case of a gene preparation, in addition to a method in which a gene encoding the protein is directly administered by injection, a method in which a vector in which the gene is incorporated is administered. Examples of the vector include an adenovirus vector, an AAV (adeno-associated virus) vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, and the like, and can be efficiently administered by using these virus vectors. Examples of non-viral vectors include liposomes, artificial lipid vesicles, hollow nanoparticles, and polymer compounds such as dendrimers.

  As the administration form of the gene preparation, local administration can be performed on the skin tissue in addition to the usual systemic administration such as intravenous and intraarterial administration.

  The dosage of the medicament of the present invention varies depending on the age, weight, symptom, dosage form, administration route, and administration frequency of the administration subject, and can vary widely. For example, an effective amount of the protein is in the range of about 0.001 to 1000 mg / kg body weight, preferably about 0.01 to 100 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight, and from once a day to several Dosage is administered for 1 day or more. In addition, when the medicament of the present invention contains a gene encoding the protein and is introduced by a method such as gene therapy, a gene capable of expressing the above range of protein may be administered.

3. Method for screening a substance that regulates the GTPase activity of Rab27A The present invention also provides a method for screening a substance that regulates the GTPase activity of Rab27A. The screening method of the present invention can be easily performed in vitro, for example, a step of contacting GTP-bound Rab27A with a test substance in the presence of the EPI64 / Rab27A-GAPα or FLJ13130 / Rab27A-GAPβ protein, A step of monitoring the GTP hydrolysis of Rab27A, and a step of selecting a test substance that promotes or suppresses GTP hydrolysis as compared with the case where the Rab27A is not contacted with the test substance.

  Since EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ proteins are GTPase-activating proteins specific for Rab27A, when they are brought into contact with GTP-bound Rab27A, GTP bound to Rab is dephosphorylated. Converted to GDP. Therefore, if the degree of this GTP hydrolysis is used as an index, it is possible to screen for substances that regulate the GTPase activity of Rab27A.

  For example, in the presence of the protein, GTP-bound Rab27A is contacted with a test substance, GTP hydrolysis is monitored, and when GTP hydrolysis is suppressed as compared with the case where the test substance is not contacted, the test substance is Rab27A -It can be selected as an inhibitor of GAP activity, that is, a candidate substance for Rab27A activation. On the other hand, when GTP hydrolysis is promoted compared to the case where the test substance is not contacted, the test substance can be selected as a substance that promotes Rab27A-GAP activity, that is, a candidate substance for inactivating Rab27A.

  The monitoring of GTP hydrolysis may be performed by the method for measuring the amount of phosphoric acid generated by the GTP hydrolysis described above.

  It is preferable that the selected candidate substance is brought into contact with melanocytes to confirm whether it induces melanosome aggregation or not, and is finally determined as a regulator of Rab27A GTPase activity. Here, “aggregation” refers to accumulation of melanosomes in the peripheral region of the melanocyte nucleus. The contact between the test substance and melanocytes is performed by culturing the melanocytes in a medium to which the test substance is added. As the melanocyte, a melanocyte derived from a mammal expressing the Rab27A gene can be used, and examples thereof include melan-a cells. Although it does not specifically limit as a mammal, A human, a mouse | mouth, a rat, etc. are preferable, Among these, a mouse | mouth is preferable.

  The type of the test substance that is the subject of the screening method of the present invention is not particularly limited. For example, a mixture containing a plurality of compounds such as extracts of animal and plant tissues or microbial cultures, and preparations purified from them; naturally occurring molecules (eg, amino acids, peptides, oligopeptides, polypeptides, proteins, Nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); or synthetic analogs or derivatives of naturally occurring molecules (eg, peptidomimetics); and non-naturally occurring molecules (eg, combinatorial chemistry techniques) Low molecular organic compounds); and mixtures thereof. In addition, as a test substance, a single test substance may be tested independently, or a test substance mixture (including a library) may be tested. Examples of the library containing a plurality of test substances include a synthetic compound library and a peptide library.

  The regulators of Rab27A GTPase activity selected by the above screening methods are melanosome transport control, hormone secretion control, digestive enzyme secretion control, immune cells (platelet, cytotoxic T It is useful for controlling granule release from cells and mast cells. Therefore, the regulatory substance is a disease associated with abnormal melanosome transport (eg, pigmentation, Griscelli syndrome, etc.), a disease associated with abnormal hormone secretion (eg, diabetes due to abnormal insulin secretion, hypoglycemia, etc.), Diseases related to abnormal digestive enzyme secretion (for example, pancreatic diseases such as acute pancreatitis, chronic pancreatitis and pancreatic cancer due to abnormal amylase secretion, salivary gland diseases, etc.), diseases related to abnormal mast cell release (eg abnormal histamine release) It can be used as an active ingredient of a medicament for the treatment and / or prevention of inflammation, allergic diseases and the like.

  More specifically, a substance that regulates the GTPase activity of Rab27A can control the transport of melanosomes from the periphery of melanocytes to the cell membrane. Therefore, Rab27A activator promotes the above melanosome transport, so it can be used, for example, as a white hair inhibitor, while Rab27A inactivator suppresses the above melanosome transport, for example, it can be used as a skin lightening agent. Can do.

  A substance that regulates the GTPase activity of Rab27A selected by the screening method can be used as an active ingredient in a composition for treating and / or preventing the above-mentioned diseases, quasi drugs, cosmetics and the like. Such a composition can be produced by a conventional method using a selected substance that controls the GTPase activity of Rab27A and an ordinary pharmaceutical carrier. For example, when an oral preparation is prepared, a binder, a disintegrant, a lubricant, a colorant and the like are added as necessary, and then a tablet, granule, powder, capsule or the like is prepared by a conventional method. When preparing a parenteral preparation, a pH adjusting agent, a buffering agent, a stabilizer, a solubilizing agent and the like are added as necessary, and an injection is prepared by a conventional method.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

The materials and methods used in the following examples are shown below.
[Materials and methods]
(a) Antibody for detection Horseradish peroxidase (HRP) -conjugated anti-FLAG tagged (M2) mouse monoclonal antibody was obtained from Sigma Chemical Co (St. Louis, MO). HRP-conjugated anti-T7 tagged mouse monoclonal antibody was obtained from Merck Biosciences Novagen (Darmstadt, Germany). Anti-Rab27A mouse monoclonal antibody and Alexa594-conjugated anti-mouse IgG goat antibody were obtained from BD Transduction Laboratories (Lexington, KY) and Molecular Probes (Eugene, OR), respectively. [α- ³² P] GTP was obtained from Amersham Biosciences (Buckinghamshire, UK).

(b) Screening target Human TBC proteins shown in Table 1 below were screened for Rab27A-GAP. For FLJ33929, KIAA0397, KIAA0608, KIAA0882, KIAA0984, KIAA1055, KIAA1171, KIAA1941, USP6, and Vrp, a truncated form containing a TBC domain was used for screening. Other TBC proteins and Rab3-GAP used full-length proteins.

(c) Plasmid construction
The cDNA encoding the open reading frame of EPI64 and FLJ13130 is derived from Marathon-Ready human brain cDNA (BD Clontech; Palo Alto, Calif.) With the following mutant oligonucleotide having a BamHI linker (underlined) or a termination codon (enclosed by a square). Each was amplified as a primer.

For EPI64:
EPI64 Met primer (sense) 5'- GGATCC ATGGCGAAGAGCAACGGAGA-3 '(SEQ ID NO: 5)
EPI64 stop primer (antisense) 5'-GGTTACAAGTAGGTGTCCTC-3 '(SEQ ID NO: 6)
For FLJ13130:
FLJ13130 Met primer (sense) 5'- GGATCC ATGTCTGGGACCTTGGAGTC-3 '(SEQ ID NO: 7)
FLJ13130 stop primer (antisense) 5'-TCAGAAGTAAGCGTCCTGCC-3 '(SEQ ID NO: 8)
PCR reaction was performed by using Ex Taq (manufactured by TAKARA) in the presence of Perfect Match PCR Enhancer (Stratagene) by repeating 40 cycles of 94 ° C for 1 minute heat denaturation, 50 ° C for 2 minutes annealing, and 72 ° C for 2 minutes. . The PCR product purified from the agarose gel by MicroSpin column (Amersham Pharmacia Biotech), followed by 1-butanol extraction and ethanol precipitation was directly inserted into pGEM-T Easy vector (Promega). Both strands were confirmed by DNA sequencing.

  Cloning of cDNAs encoding other TBC proteins and Rab3-GAP was also performed according to the above procedure except for the design of primers.

  PEF- modified from TBC protein cDNA from pEGFP-C1 vector (BD Biosciences Clontech, Palo Alto, CA) and pEF-BOS (Mizushima, S., and Nagata S. 1990. Nucleic Acids Res. 18: 5322.) T7 expression vector or pEF-T7-GST expression vector (Fukuda, M. et al. 1994. J. Biol. Chem. 269: 29206-29211 .; Fukuda, M. et al. 1999. J. Biol. Chem. 274 : 31421-31427 .; Fukuda, M. et al. 2002. J. Biol. Chem. 277: 12432-12436.).

  Two EPI64 mutants, a mutant in which arginine at amino acid position 160 is replaced with lysine (referred to as “EPI64-R160K”), and a mutant in which glutamic acid at amino acid position 157 is replaced with alanine (referred to as “EPI64-D157A”) Is described in the literature (Fukuda, M. et al. 1995. J. Biol. Chem. 270: 26523-26527.) Using the following mutant oligonucleotide having an artificial XhoI site (underlined) as a primer. As described above, it was produced by a two-step PCR method.

For EPI64-R160K:
TBC primer (sense) 5'-TGTTTGTGT CTCGAG GGGGC-3 '(SEQ ID NO: 9)
R160K primer (antisense) 5'-GCCCC CTCGAG ACACAAACATCTCATGGAATGGGAACTGCTTGTGCAG-3 '(SEQ ID NO: 10)
For EPI64-D157A:
TBC primer (sense) 5'-TGTTTGTGT CTCGAG GGGGC-3 '(SEQ ID NO: 9)
D157A Primer (antisense) 5'-GCCCC CTCGAG ACACAAACATCTCATGGAATGGGAACTGCCGGTGCAGGGCACGCTC-3 '(SEQ ID NO: 11)
Thereafter, the mutant EPI64 fragment was subcloned into a modified pEF-T7 expression vector or pEGFP-C1 vector.

  pEGFP-C1-EPI64 + A (added alanine to the C-terminus of EPI64) and pEGFP-C1-EPI64-Δ449 (deleted 59 C-terminal amino acids) were similarly constructed by a conventional PCR method.

  Other expression plasmids (pEF-FLAG-Rab27A, pGEX4T-3-Rab1A, pGEX4T-3-Rab3A, pGEX4T-3-Rab4A, pGEX4T-3-Rab5A, pGEX4T-3-Rab27A, and pEF-T7-GST-Slac2- a-SHD) is described in the literature (Fukuda, M. et al. 2002. J. Biol. Chem. 277: 12432-12436 .; Kuroda, TS et al. 2002. J. Biol. Chem. 277: 9212-9218. ).

(d) Screening of TBC protein capable of inducing melanosome aggregation in melanocytes Immortalized melanocyte cell line melan-a (contributed by Dorothy C. Bennett) literature (Bennett, DC et al. 1987. Int. J. Cancer 39 : 414-418 .; Kuroda, TS et al. 2003. Mol. Cell. Biol. 23: 5245-5255.). Transfection of pEGFP-C1-TBC proteins (eg, pEGFP-C1-EPI64) into melan-a cells using FuGENE 6 (Roche Molecular Biochemicals) and melanosome aggregation assays are described in the literature (Kuroda, TS et al., ibid. ).

  The fluorescence of the cells was examined by a bright field image using a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). The distribution of melanosomes in transfected cells (“aggregation” means that melanosomes in normal melan-a cells are localized around the cells as shown in the upper right panel of FIG. As shown in the lower panel, melanosome accumulation around the nucleus) was evaluated as described in the literature (Kuroda, TS et al. Ibid.).

(e) GTP-Rab27A pull-down assay The plasmid was transfected into COS-7 cells (7.5 × 10 ⁵ cells / 10 cm-dish, the day before transfection) using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. COS-7 cells expressing FLAG-tagged Rab27A (FLAG-Rab27A) and T7-tagged TBC protein were lysed with buffer [50 mM HEPES-KOH (pH 7.2), 150 mM NaCl, 10% glycerol, 50 μM PMSF, 10 μM leupeptin , And 5 μM pepstatin A] and then solubilized with 1% Triton-X 100. After centrifugation, the supernatant was appropriately diluted with a lysis buffer so that the amount of FLAG-Rab27A protein in the diluted sample was equal when immunoblotting with an HRP-conjugated anti-FLAG tagged antibody. The diluted sample was added to the T7-GST-tagged SHD region of Slac2-a [T7-GST-Slac2-a-SHD: GTP-Rab27A capture agent (Fukuda, M. 2002. J. Biol. Chem. 277: 40118-40124 .)] Bound to glutathione-Sepharose beads (Amersham Biosciences) for 1 hour at 4 ° C. After washing the beads three times, GTP-Rab27A trapped on the beads was analyzed by 10% SDS-PAGE, followed by HRP-conjugated anti-FLAG tagged antibody (1/5000 dilution) or HRP-conjugated anti-T7 tagged antibody (1 Immunoblotting was performed at a dilution of 5,000.

(f) Purification of GST-Rab and in vitro GAP assay
Rab GST-fusion protein is expressed in E. coli JM109 and purified by standard protocols (Kuroda, TS, and Fukuda M. 2005. Methods Enzymol. 403: 431-444.) And bovine serum albumin (BSA) is standard The protein concentration was measured using a Bio-Rad protein assay kit (Hercules, CA). The in vitro GAP activity of EPI64 and FLJ13130 was measured with a slight improvement in the literature (Otomo, A. et al. 2003. Hum. Mol. Genet. 12: 1671-1687.). That is, GST-Rab27A produced by E. coli incubated with [α- ³² P] GTP was passed through a Sephadex G-25 column to eliminate unbound GTP. The GAP reaction is initiated by adding 2 pmol of TBC protein or BSA (control) to a solution containing 2 pmol of either Rab1A, Rab3A, Rab4A, Rab5A, or Rab27A protein and incubating at 30 ° C. did. Since the intrinsic GTPase activity of Rab family members is different, the reaction time was changed depending on the Rab isoform tested (Rab1A, 1 hour; Rab3A, 15 minutes; Rab4A, 1 hour; Rab5A, 12 minutes; Rab27A, 1 hour) . The reaction was stopped by the addition of 20 mM EDTA and 0.4% SDS. 2 μl of sample was placed in a TLC plate (Merck Biosciences, Darmstadt, Germany) and developed with 0.5 M LiCl and 1 M formic acid. The amounts of GTP and GDP were measured with a BAS-2500 Bioimage analyzer (FUJIFILM, Tokyo, Japan) using an imaging plate.

(g) Immunostaining of melanocytes
The fixation of melan-a cells and immunostaining with endogenous Rab27A protein were performed as described in the literature (Kuroda, TS et al. 2003. Mol. Cell. Biol. 23: 5245-5255.).

(h) Sequence analysis
Multiple sequence alignment and phylogenetic analysis of TBC protein was performed using the CLUSTAL_X program (Thompson, JD et al. 1997. Nucleic Acids Res. 25: 4876-4882.). Following the amino acid sequence and the position of the TBC domain in the NCBI database, a phylogenetic tree was created (FIG. 3C).

[result]
(Example 1) Identification of Rab27A-GAP
(1) Comprehensive screening of putative Rab27A-GAP using melanosome aggregation in melanocytes as an indicator
Targeting 40 different human TBC / Rab-GAP proteins (see Table 1 above), each GFP (green fluorescent protein) -tagged TBC protein was cultured in cultured melanocytes for primary screening of Rab27A-GAP. It was overexpressed in certain melan-a cells (Bennett, DC et al. 1987. Int. J. Cancer 39: 414-418.).

As shown in the top panel on the right side of FIG. 1, normal melan-a cells (cells expressing only GFP) were elongated or dendritic, and melanosomes were localized around the cell membrane. . If the TBC protein inactivates Rab27A by promoting its GTPase activity, overexpression of the TBC protein may occur in patients with Griscelli syndrome or the corresponding mouse model ashen (Menasche, G. et al. 2000 Nat. Genet. 25: 173-176 .; Wilson, SM et al. 2000. Proc. Natl. Acad. Sci. USA 97: 7933-7938. It is expected that.

  As expected, of the 40 TBC proteins tested, two of EPI64 and FLJ13130 strongly induced typical melanosome aggregation, and FLJ22474 and Vrp appeared to be associated with weak perinuclear aggregation ( See Table 1 above). Melan-a cells expressing GFP-tagged FLJ22474 (GFP-FLJ22474), GFP-Vrp, GFP-EPI64, or GFP-FLJ13130 showed melanosome aggregation but had no effect on cell morphology ( Figure 1, left, bottom four panels). GFP-Vrp, GFP-EPI64 and GFP-FLJ13130 were localized around the cell, whereas GFP-FLJ22474 was localized in the cytoplasm.

  Of the above four TBC proteins, FLJ22474 is an uncharacterized protein and Vrp has been reported to be involved in angiogenesis (Yonekura, H. et al. 2001. Ann. NY Acad. Sci. 947: 382-386.) EPI64 is also characterized as a binding protein of EBP50 that functions for actin bundling around the cell, but EPI64 itself may not be involved in actin bundling (Reczek, D., and Bretscher A. 2001. J. Cell Biol. 153: 191-206.). FLJ13130 is a close homologue of EPI64, but nothing is known about its function or localization. However, there are no reports on the GAP activity of these four TBC proteins.

  Members of the Rab27 and Rab3 subfamilies are closely related in the Rab family phylogenetic tree and share several effectors (eg, rabphilin and Noc2) (Fukuda, M. 2003. J Biol. Chem. 278: 15373-15380 .; Fukuda, M. et al. 2004. J. Biol. Chem. 279: 13065-13075.), Rab3 established to be a GAP of Rab3 without the TBC domain -GAP may also act as Rab27A-GAP. However, even when GFP-tagged Rab3-GAP was expressed in melan-a cells, Rab3-GAP did not induce any melanosome aggregation (Table 1 above). This suggests that a specific Rab27A-GAP exists for Rab27A.

(2) Secondary screening of putative Rab27A-GAP by GTP-Rab27A pull-down assay To further investigate whether the above four TBC proteins induce melanosome aggregation by directly inactivating Rab27A on melanosomes A GTP-Rab27A pull-down assay was performed using Slac2-a SHD. Slac2-a SHD specifically recognizes GTP-bound Rab27A and not other GTP-Rabs (Fukuda, M. 2002. J. Biol. Chem. 277: 40118-40124 .; Fukuda, M. 2003. J. Biol. Chem. 278: 15373-15380 .; Kuroda, TS et al. 2002. J. Biol. Chem. 277: 9212-9218.). Therefore, the amount of active Rab27A in the cell lysate can be calculated by quantitatively measuring Rab27A bound to the Slac2-a SHD column (ie, GTP-Rab27A pull-down assay).

  Specifically, the GTP-Rab27A pull-down assay was performed as follows. First, COS-7 cell lysate containing FLAG-Rab27A and T7-tagged TBC protein was incubated with glutathione-Sepharose beads conjugated with purified T7-GST-Slac2-a-SHD. Four TBC proteins (EPI64, FLJ13130, Vrp, and FLJ22474) confirmed to induce melanosome aggregation as TBC proteins, and three TBC proteins (MGC34741, Usp6, and Prc17) that have no effect on melanosome distribution as negative controls Was used. GTP-Rab27A protein captured on whole cell lysate (Input) and SHD column (IP) is treated with 10% SDS-PAGE and HRP-conjugated anti-FLAG tagged antibody (Figure 2, second panel from top) or HRP-conjugated anti-antibody Analysis was by immunoblotting using a T7 tagged antibody (Figure 2, bottom two panels). The lysate (1/60 of the reaction mixture) used as input was analyzed in the same manner using an HRP-conjugated anti-FLAG tagged antibody (FIG. 2, top panel).

  When Rab27A was co-expressed with EPI64 or FLJ13130, no active Rab27A was visually detected under this test condition (FIG. 2, second panel from the top, lanes 2 and 3). In contrast, when Rab27A was co-expressed with FLJ22474 or Vrp, the amount of active Rab27A was almost affected, as was the case with co-expression with negative control MGC34741, Usp6, or Prc17. No, or slightly decreased (Figure 2, second panel from top, lanes 4 and 5).

  The above GTP-Rab27A pull-down assay results were in good agreement with the results obtained by primary screening of putative Rab27A-GAP by melanosome aggregation assay. EPI64 and FLJ13130 both induced melanosome aggregation in melanocytes and actually reduced the amount of GTP-Rab27A when co-expressed with Rab27A. In contrast, FLJ22474 and Vrp, which induced a weak aggregation phenotype in melanocytes, had little effect on the amount of GTP-Rab27A. This suggests that these two TBC proteins induce partial melanosome aggregation by a mechanism different from GTP-Rab27A down-regulation.

  Based on the above results, EPI64 and FLJ13130 were selected as Rab27A-GAP candidates from TBC protein family members and named EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ, respectively.

(Example 2) Function prediction based on the structures of EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ In a database search, EPI64 / Rab27A-GAPα, FLJ13130 / Rab27A-GAPβ, and FLJ00332 (not inducing melanosome aggregation: see Table 1) ) Has been shown to form a subfamily of TBC proteins (FIG. 3C, shaded area). These three proteins have the same domain structure (a TBC domain in the central region and a specific sequence at the C-terminus that interacts with the PDZ domain (DTYL in EPI64, DAYF in FLJ13130, and DTRF in FLJ00332); Reference]] (Kornau, HC et al. 1997. Curr. Opin. Neurobiol. 7: 368-373 .; Reczek, D., and Bretscher A. 2001. J. Cell Biol. 153: 191-206 .; Saras, J., and Heldin CH 1996. Trends Biochem. Sci. 21: 455-458.). Their overall sequence is highly similar (61.9% identity between EPI64 and FLJ13130, 50.0% between EPI64 and FLJ00332, 48.9% identity between FLJ13130 and FLJ00332), especially in the TBC domain (81.1% between EPI64 and FLJ13130, 60.2% between EPI64 and FLJ00332, 59.2% between FLJ13130 and FLJ00332; see FIGS. 3A and B). All three of these proteins are yeast Rab-GAP, Gyp1 (Albert, S. et al. 1999. EMBO J. 18: 5216-5225.) (Asterisk in FIG. 3B) and several mammalian TBC protein GAPs. Because it has a conserved arginine that has been shown to be essential for activity (Gao, XD et al. 2003. J. Cell Biol. 162: 635-646 .; Lanzetti, L. et al. 2000. Nature 408: 374-377 .; Pei, L. et al. 2002. Cancer Res. 62: 5420-5424.), Which may have some Rab-GAP activity. Since the sequence of the TBC domain of FLJ00332 is slightly different from the sequence of the TBC domain of EPI64 or FLJ13130, FLJ00332 is thought to function as a GAP of Rab (eg, Rab3A, Rab26, or Rab37) related to Rab27A.

Example 3 In vitro Rab27A-GAP activity of EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ
In order to verify whether EPI64 / Rab27A-GAPα and FLJ13130 / Rab27A-GAPβ actually function as Rab27A-GAP, purified recombinant EPI64 (T7-GST-EPI64), FLJ13130 (T7-GST-FLJ13130) In vitro Rab27A (GST-Rab27A) GAP activity was measured.

  Consistent with the results of the melanosome aggregation assay (Figure 1) and the GTP-Rab27A pull-down assay (Figure 2), the purified recombinant EPI64 and FLJ13130 actually activated the RTP27A GTPase activity slightly but specifically. (FIG. 4B), EPI64 GAP activity appeared to be stronger than that of FLJ13130 (FIG. 4A). In contrast, the negative control Rab3-GAP did not show any Rab27A-GAP activity. Some previously characterized TBC proteins (eg, GAP-CenA and RN-Tre) have relatively broad specificity (Cuif, MH et al. 1999. EMBO J. 18: 1772- 1782 .; Haas, AK et al. 2005. Nat. Cell Biol. 7: 887-893), EPI64 and FLJ13130 may also exert GAP activity against other Rabs, so Rab-GAP of EPI64 Specificity was examined. However, recombinant EPI64 did not show any GAP activity against the other Rab tested (Rab1A, 3A, 4A, 5A, 10) (FIG. 4C). In particular, EPI64 lacked GAP activity against Rab3A, the closest isoform of Rab27A. This strongly suggests that EPI64 is probably a TBC protein that also functions as FLJ13130 as a specific Rab27A-GAP.

(Example 4) Identification of Rab27A-GAP active domain of EPI64
Although EPI64 and FLJ13130 actually demonstrated Rab27A-GAP activity by two screening methods, EPI64 and FLJ13130's own TBC domain has Rab27A-GAP activity and is induced by EPI64 or FLJ13130 It remains unknown whether melanosome aggregation is due to inactivation of Rab27A in living cells.

  To elucidate the above matters, a mutant of EPI64 (referred to as EPI64-R160K) in which conservative arginine necessary for GAP activity is substituted with lysine, or a mutant of EPI64 in which conservative glutamic acid is substituted with alanine (EPI64- D157A) is described in a previous research report (Gao, XD et al. 2003. J. Cell Biol. 162: 635-646 .; Lanzetti, L. et al. 2000. Nature 408: 374-377.) It was constructed by the method.

  A GTP-Rab27A pull-down assay was performed in the same manner as in Example 1. COS-7 cells expressing FLAG-Rab27A only, FLAG-Rab27A and T7-EPI64, FLAG-Rab27A and T7-EPI64-R160K, and FLAG-Rab27A and T7-EPI64-D157A, respectively, were purified from T7-GST-Slac2- Incubated with glutathione-Sepharose beads conjugated with a-SHD. Total lysate (FIG. 5A: Input, lanes 1-4) and protein captured by the SHD region (FIG. 5A: IP, lanes 5-8) were 10% SDS-PAGE and HRP-conjugated anti-FLAG tagged antibody (FIG. 5A). , Top panel) or by immunoblotting with HRP-conjugated anti-T7 tagged antibody (FIG. 5A, bottom two panels).

  In the GTP-Rab27A pull-down assay in COS-7 cells, wild-type EPI64 dramatically decreased the amount of active Rab27A, whereas neither EPI64-R160K nor EPI64-D157A decreased the amount of active Rab27A. (See the top panel of FIG. 5A, see lanes 6-8). It was not due to the low expression level of the recombinant protein in COS-7 cells that the two EPI64 mutants lost their effect. This is because they are expressed much more stably than wild type EPI64 (middle panel of FIG. 5A, lanes 2-4). From the above, it was concluded that the TPI domain of EPI64 is a functional Rab27A-GAP domain.

  The phenotype of melan-a cells expressing EPI64 mutants (GFP-EPI64-R160K and GFP-EPI64-D157A) was in complete agreement with the results of the GTP-Rab27A pull-down assay (FIG. 5B). Both EPI64-R160K and EPI64-D157A mutants, as well as GFP alone, lost the ability to fully induce melanosome aggregation (FIG. 5C). It should be noted that both EPI64 mutants, like wild-type EPI64, are localized around the cell (FIG. 5B), indicating that both mutants (R160K and D157A) lose only Rab27A-GAP activity. , Suggesting that other properties of the EPI64 protein are retained.

Example 5 EPI64 Melanosome Aggregation by Rab27A Inactivation
EPI64 has been shown to localize to the periphery of cells through interaction with EBP50 involved in actin bundling (Reczek, D., and Bretscher A. 2001. J. Cell Biol. 153: 191-206 .). It is also known that interference with actin remodeling by the C-terminal region of Slac2-a induces melanosome aggregation (Fukuda, M., and Itoh T. 2004. J. Biol. Chem. 279: 22314- 22321 .; Kuroda, TS et al. 2003. Mol. Cell. Biol. 23: 5245-5255.). From these facts, it can be said that EPI64 can also influence the melanosome distribution by causing excessive actin bundling through interaction with EBP50 in addition to inactivation of Rab27A. To eliminate this possibility, two C-terminal mutants, EPI64 + A and EPI64-Δ449 (see FIG. 6A), were created [none of these mutants interact with EBP50 (Reczek, D., and Bretscher A. 2001. J. Cell Biol. 153: 191-206.)]. As expected, when overexpressed in melan-a cells, both GFP-tagged mutants induced melanosome aggregation, similar to wild-type EPI64 (FIG. 6B). These results suggest that induction of melanosome aggregation by EPI64 relies on inactivation of Rab27A function rather than excessive actin filament bundling. The EPI64 C-terminal mutant, like EPI64 wild type, can target the periphery of actin-rich cells (FIG. 6B), so that placental cells (Reczek, D., and Bretscher A. 2001. J. Cell Biol. 153: 191 In contrast to -206.), Other factors than EBP50 were thought to determine the localization of EPI64 in melanocytes.

Example 6 Rab27A exclusion on melanosomes by expression of EPI64 in melanocytes In general, GDP-bound inactive Rab is extracted from the membrane by GDP-dissociation inhibitor (GDI) for recycling of Rab proteins (Seabra , MC, and Wasmeier C. 2004. Curr. Opin. Cell Biol. 16: 451-457.). Therefore, GDP-bound Rab27A inactivated by EPI64 was also expected to be extracted from the melanosome membrane.

  After melan-a cells transfected with a vector encoding GFP-EPI64 were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.3% Triton X-100, they were anti-Rab27A antibody and Alexa594-conjugated anti-mouse. Stained with IgG antibody. Cells were examined for Rab27A fluorescence and GFP-EPI64 fluorescence by confocal microscopy.

As shown in FIG. 7, Rab27A is present on melanosomes of untransfected cells (arrowheads), but no Rab27A signal was detected on melanosomes in GFP-EPI64 expressing cells (arrows).
Therefore, it was concluded that EPI64 inactivates Rab27A in vivo by promoting the GTPase of Rab27A.

Shows GFP fluorescence image (left column) and corresponding bright field image (right column) of melan-a cells expressing Rab27A-GAP (TBC protein) candidate [GFP only (top panel), GFP Melan-a cells expressing -FLJ22474 (second panel from the top), GFP-Vrp (middle panel), GFP-EPI64 (second panel from the bottom), and GFP-FLJ13130 (bottom panel) ]. Arrows indicate cells expressing each GFP-tagged TBC protein and representing melanosome aggregation. Bar: 20 μm. FIG. 2 shows the results of GTP-Rab27A pull-down assay [FLAG-Rab27A only (control; lane 1), FLAG-Rab27A + T7-EPI64 (lane 2), FLAG-Rab27A + T7-FLJ13130 (lane 3), FLAG-Rab27A + T7-FLJ22474 (Lane 4), FLAG-Rab27A + T7-Vrp (lane 5), FLAG-Rab27A + T7-MGC34741 (lane 6), FLAG-Rab27A + T7-Usp6 (lane 7), FLAG-Rab27A + T7-Prc17 (lane 8)]. Asterisk (*) indicates TBC protein that induced melanosome aggregation in melan-a cells. FIG. 3A shows a schematic diagram of EPI64 / Rab27A-GAPα, FLJ13130 / Rab27A-GAPβ, and FLJ00332. The TBC domain is indicated by a gray box and the last four amino acids (ie, the putative recognition sequence by the PDZ domain) are shown on the right side of the diagram. The percentage of amino acid identity of the TBC domain that the two proteins have in common is shown on the right. FIG. 3B shows the sequence alignment of the TBC domains of EPI64, FLJ13130, and FLJ00332. The sequences of the TBC domains of EPI64 (amino acid residues 108-313), FLJ13130 (82-287), and FLJ00332 (89-294) were aligned with the CLUSTAL_X (version 1.8) program. Conserved amino acids are shown in gray background. An asterisk (*) indicates a conserved residue essential for the catalytic activity of the Gyp1 GAP domain. The black arrowhead indicates the amino acid residue substituted in the EPI64 mutant (EPI64-D157A and EPI64-R160K). The number of amino acids is shown at the end of each sequence. FIG. 3C shows a phylogenetic tree of human TBC domains. The phylogenetic tree was created using the CLUSTAL_X (version 1.8) program. EPI64, FLJ13130, and FLJ00332 form a small branch (ie, a subfamily of TBC proteins; enclosed in gray). FIG. 4A shows the GAP activity of EPI64, FLJ13130, and Rab3-GAP. FIG. 4B shows the results of observation over time of GTP hydrolysis of Rab27A in the presence of BSA or EPI64. The results were expressed as a percentage of the amount of GTP-bound Rab27A after the reaction relative to the amount of GTP-bound Rab27A before the reaction. Bars represent the mean ± standard deviation of data from three independent tests. *: P <0.05 (Student's unpaired t test) FIG. 4C shows the Rab-GAP specificity of EP164 [Rab1A (1 hour), Rab3A (15 minutes), Rab4A (1 hour), Rab5A (12 minutes), and Rab-GAP activity of EPI64 against Rab27A (1 hour)]. GTP activity of each Rab protein in the presence of BSA (white bar) or EPI64 (black bar) is shown. *: P <0.05 (Student's unpaired t test) FIG. 5A shows the results of the GTP-Rab27A pull-down assay [FLAG-Rab27A alone (control; lanes 1, 5), FLAG-Rab27A + T7-EPI64 (lanes 2, 6), FLAG-Rab27A + T7-EPI64-R160K (lanes 3, 5). 7), FLAG-Rab27A + T7-EPI64-D157A (lanes 4, 8)]. FIG. 5B shows a GFP fluorescence image (right column) of melan-a cells transfected with a vector encoding GFP-EPI64 (upper), GFP-EPI64-R160K (middle), or GFP-EPI64-D157A (lower). Indicates. The bright field image (left column) shows the melanosome distribution within the cell. Arrowheads indicate melan-a cells that exhibit melanosome aggregation. Bar: 20 μm. FIG. 5C shows a summary of the results of the melanosome distribution assay. Images of the transfected cells are randomly captured using GFP fluorescence as an index, and whether or not melanosomes aggregate around the nucleus based on the corresponding bright field image (Kuroda, TS et al. 2003. Mol. Cell. Biol. 23: 5245-5255.). Results were expressed as the percentage of cells that showed melanosome aggregation around the nucleus, and the values were the mean ± standard deviation of data from three independent experiments (n> 150). *: P <0.05 (Student's unpaired t test) FIG. 6A shows a schematic diagram of a C-terminal mutant of EPI64. EPI64 + A contains an additional alanine residue at the C-terminus that eliminates interaction with EBP50. EPI64-Δ449 has 59 amino acids at the C-terminal deleted. FIG. 6B shows typical images of cells expressing wild-type EPI64 or C-terminal mutant of EPI64 [GFP-EPI64 (upper), GFP-EPI64 + A (middle), GFP-EPI64-Δ449 (lower). Expressing melan-a cells]. GFP fluorescence is shown on the left and the corresponding bright field image is shown on the right. Bar: 20 μm. Fluorescence images of melan-a cells transfected with a vector encoding GFP-EPI64 are shown [Rab27A fluorescence (red on the left) and GFP-EPI64 fluorescence (green in the middle)]. The bright field image (right) shows the melanosome distribution in the cells. Bar: 20 μm.

Claims (5)

A Rab27A inactivating agent containing any one of the following (a) to (d):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Rab27A Protein with GTPase activity promoting action
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and Rab27A Protein with GTPase activity promoting action
(c) a gene encoding the protein of (a) or (b)
(d) a recombinant vector comprising a gene encoding the protein of (a) or (b)   A medicament for treating and / or preventing a disease associated with Rab27A activation, comprising the Rab27A inactivating agent according to claim 1 as an active ingredient.   The medicament according to claim 2, wherein the disease accompanied by Rab27A activation is pigmentation. A method for screening a substance that regulates the GTPase activity of Rab27A, comprising the following steps.
(1) A step of bringing GTP-bound Rab27A into contact with a test substance in the presence of any of the following proteins (a) or (b)
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Rab27A Protein with GTPase activity promoting action
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and Rab27A Protein with GTPase activity promoting action
(2) Process for monitoring RTP27A GTP hydrolysis
(3) A step of selecting a test substance that promotes or suppresses GTP hydrolysis compared to the case where the test substance is not contacted.   The method according to claim 4, wherein the control of Rab27A GTPase activity is melanosome transport control, hormone secretion control, digestive enzyme secretion control, or granule release control from immune cells.

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