WO2014104224A1 - PGC-1β蛋白質の機能調整剤,ミトコンドリア機能の調節剤,抗肥満剤及びそれらスクリーニング方法 - Google Patents
PGC-1β蛋白質の機能調整剤,ミトコンドリア機能の調節剤,抗肥満剤及びそれらスクリーニング方法 Download PDFInfo
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Definitions
- the present invention relates to a PGC-1 ⁇ protein function regulator, a mitochondrial function regulator, an anti-obesity agent, and a screening method thereof.
- PPARs Peroxisome proliferator-activated receptors
- PGC-1 ⁇ PGC-1 ⁇
- PGC-1-related coactivators PRC
- PGC-1 ⁇ is known to be involved in brown fat determination and / or differentiation, cell metabolism, fatty acid oxidation, mitochondrial function and / or respiration (see Patent Document 1).
- PGC-1 ⁇ is known to be involved in the regulation of lipid biosynthesis and lipid transport (see Patent Document 2).
- Pgc-1-related coactivator a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells, Mol Cell Biol. 21, 3738-3749. Kressler, D. et al., (2002).
- the PGC-1-related protein PERC is aselective coactivator of estrogen receptor alpha. J Biol Chem 277, 13918-13925. Lin, J. (2001).
- PGC-1beta Peroxisome Proliferator-activated Receptor gammaCoactivator 1beta
- PGC-1beta Peroxisome Proliferator-activated Receptor gammaCoactivator 1beta
- PGC-1alphaand PGC-1beta Bioenergetic analysis of peroxisomeproliferator-activated receptor gamma coactivators 1alpha and 1beta
- PPARgamma coactivator 1beta / ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditureand antagonizes obesity.
- PPAR coactivator 1 (PGC-1) ⁇ has various functions. Therefore, it is desired to provide a substance that enhances the activity of PGC-1 ⁇ and a method for screening such a substance.
- An object of the present invention is to provide a substance that enhances the activity of PGC-1 ⁇ and a method for screening such a substance.
- An object of the present invention is to provide a modulator of mitochondrial function that is effective in the treatment or prevention of obesity and a screening method thereof.
- the inventors of the present invention have found that a conditional knockout mouse for Synoviolin significantly upregulates a gene group involved in ⁇ -oxidation and / or mitochondrial biogenesis, and that the number of mitochondria is markedly increased in white adipocytes.
- the present inventors found for the first time that the coactivator PGC-1 ⁇ , which regulates the transcription of RNA, is negatively regulated by Synoviolin.
- the first aspect of the present invention relates to a function regulator of PGC-1 ⁇ protein (the agent of the present invention).
- This function regulator of PGC-1 ⁇ protein contains a synoviolin expression inhibitor or a synoviolin activity inhibitor as an active ingredient.
- the function regulator of PGC-1 ⁇ protein promotes fatty acid ⁇ oxidation and promotes mitochondrial expression or activity by adjusting the activity of PGC-1 ⁇ protein, for example.
- the function regulator of the PGC-1 ⁇ protein of the present invention is effective for the treatment and prevention of symptoms such as obesity involving the PGC-1 ⁇ protein.
- the agent of the present invention can increase the activity of the PGC-1 ⁇ protein by inhibiting synoviolin, thereby increasing the activity of mitochondria, and thus is useful for the prevention or treatment of obesity.
- a preferred embodiment of the function regulator of PGC-1 ⁇ protein according to the first aspect of the present invention is used for promoting fatty acid ⁇ oxidation by PGC-1 ⁇ protein and / or promoting or promoting mitochondrial expression or activity It is.
- the PGC-1 ⁇ protein function regulator of the present invention is preferably used for mitochondrial activation. Therefore, the present invention also provides a mitochondrial activator. This mitochondrial activator is used, for example, to cause either or both of increased mitochondrial expression and increased mitochondrial size.
- the second aspect of the present invention relates to a method of screening for a PGC-1 ⁇ protein function regulator.
- a test substance is allowed to act on cells of an adipose tissue or an animal individual.
- at least one of the expression level of synoviolin in cells of adipose tissue, the binding between synoviolin and PGC-1 ⁇ protein, and the ubiquitination of PGC-1 ⁇ protein by synoviolin is measured or detected.
- the present invention is based on the finding that inhibiting the activity of Synoviolin leads to an increase in the activity of PGC-1 ⁇ protein. Therefore, by evaluating the expression and activity of Synoviolin, it is possible to screen for a function regulator of PGC-1 ⁇ protein (a substance having an action of regulating the function of PGC-1 ⁇ protein).
- An embodiment of the second aspect of the present invention relates to a method for detecting (screening) a mitochondrial activator using the above-described method for screening a function regulator of PGC-1 ⁇ protein.
- An embodiment of the second aspect of the present invention relates to a method for detecting (screening) a therapeutic or prophylactic agent for obesity using the method for screening a function regulator of PGC-1 ⁇ protein described above.
- the present invention can provide, for example, a PGC-1 ⁇ protein function regulator and a screening method thereof, a mitochondrial function regulator effective in the treatment or prevention of obesity, and a screening method thereof.
- FIG. 1 is a graph showing ⁇ -oxidation and transcription levels of mitochondria-related genes in white adipocytes derived from Synoviolin knockout mice.
- FIG. 2 is an electron micrograph of mitochondria in white adipocytes derived from Synoviolin knockout mice. The left-hand photo is a wild-type mouse (syno WT) as a control, and the right-hand side is a synoviolin knockout mouse (synocKO). The magnification of the electron microscope is 5,000 times.
- FIG. 3 is a Western blot showing the binding of PGC-1 ⁇ and Synoviolin in vitro.
- FIG. 4 is a conceptual diagram of PGC-1 ⁇ and fragmented PGC-1 ⁇ and a Western blot showing the binding of fragmented PGC-1 ⁇ and Synoviolin in vitro.
- the abbreviations in Fig. 4 are as follows. AD: activation domain, LXXLL: LXXLL motif, E: glutamate rich domain, SR: serine-arginine rich domain, RRM: RNA recognition motif.
- FIG. 5 is a conceptual diagram of Synoviolin and fragmented Synoviolin and a Western blot showing the binding between PGC-1 ⁇ and fragmented Synoviolin in vitro. The abbreviations in FIG.
- FIG. 6A is a Western blot showing the binding of PGC-1 ⁇ and Synoviolin in vivo.
- FIG. 6B is another western blot showing binding of PGC-1 ⁇ and Synoviolin in vivo.
- FIG. 7 is a fluorescence micrograph showing the localization of PGC-1 ⁇ and Synoviolin.
- FIG. 8A is a Western blot showing ubiquitination of PGC-1 ⁇ by Synoviolin in vitro.
- FIG. 8B is a Western blot showing ubiquitination of PGC-1 ⁇ by Synoviolin in vivo.
- FIG. 8A is a Western blot showing ubiquitination of PGC-1 ⁇ by Synoviolin in vitro.
- FIG. 8B is a Western blot showing ubiquitination of PGC-1 ⁇ by Synoviolin in vivo.
- FIG. 8C is a Western blot showing the effect of in vivo synoviolin knockout on the protein level of PGC-1 ⁇ .
- FIG. 8D is a Western blot showing the effect of Synoviolin knockout in vivo on PGC-1 ⁇ proteolysis.
- FIG. 9A is a Western blot showing the effect of Synoviolin siRNA on PGC-1 ⁇ protein expression.
- FIG. 9B is a Western blot showing the effect of Synoviolin siRNA on functional expression of PGC-1 ⁇ .
- FIG. 9C is a graph showing the influence of overexpression of Synoviolin on the functional expression of PGC-1 ⁇ .
- FIG. 9D is an electron micrograph showing changes in mitochondria morphology caused by Synoviolin siRNA.
- FIG. 9A is a Western blot showing the effect of Synoviolin siRNA on PGC-1 ⁇ protein expression.
- FIG. 9B is a Western blot showing the effect of Synoviolin siRNA on functional expression of PGC-1 ⁇ .
- FIG. 9C is
- FIG. 10 is a Western blot showing the inhibitory effect of synoviolin binding to synoviolin and PGC-1 ⁇ by an E3 ubiquitin ligase activity inhibitor.
- FIG. 11-1 is a Western blot showing the binding inhibitory effect between Synoviolin and PGC-1 ⁇ by the test substances 349 and 348 (upper figure), and a graph evaluating the binding ability of the test substances 349 and 348 (lower figure).
- FIG. 11-2 is a Western blot showing the binding inhibitory effect between Synoviolin and PGC-1 ⁇ by the test substances quercetin and 351 (upper figure), and a graph evaluating the binding ability of the test substances quercetin and 351 (lower figure left and right) It is.
- FIG. 11-1 is a Western blot showing the binding inhibitory effect between Synoviolin and PGC-1 ⁇ by the test substances 349 and 348 (upper figure), and a graph evaluating the binding ability of the test substances 349 and 348 (lower figure left and right) It is.
- FIG. 11-3 is a Western blot showing the binding inhibitory effect of each test substance.
- FIG. 11-4 is another Western blot showing the inhibitory effect on the binding between Synoviolin and PGC-1 ⁇ by the test substance. The degree of binding was expressed in a graph as intensity after analyzing the image of a Western blot subjected to the binding assay.
- FIG. 12 is a graph showing the effect of the test substance on the PGC-1 ⁇ function.
- FIG. 13 is an electron micrograph showing changes in mitochondrial morphology caused by the test substance.
- FIG. 14 is a graph showing oxygen consumption of adipocytes in Synoviolin knockout mice.
- FIG. 15 is a graph showing the amount of basal metabolism in Synoviolin knockout mice.
- FIG. 16 is a photograph replacing a drawing showing Western blotting of adiponectin and synoviolin in each tissue of wild type WT and synoviolin KO.
- FIG. 17 shows a Western blot for measuring the half-life of PGC-1 ⁇ .
- FIG. 18 is an electron micrograph showing the mitochondrial morphology change in adipose tissue by LS-102.
- FIG. 19 is a Western blotting showing the half-life of PGC-1 ⁇ .
- FIG. 20 shows the results of an in vitro ubiquitination assay.
- FIG. 21 is a western blot showing the concentration dependency of the ubiquitination inhibitory activity of PGC-1 ⁇ by the test substances 348 and 349.
- FIG. 22 is the amino acid sequence of the SyU domain in multiple species.
- FIG. 23 is a Western blot of SYVN1 SyU mutant.
- FIG. 24 is a Western blot of mutants of SYVN 1266-270aa.
- the first aspect of the present invention relates to a function regulator of PGC-1 ⁇ protein (the agent of the present invention).
- PGC-1 ⁇ means PPAR coactivator 1 ⁇ .
- the function regulator for PGC-1 ⁇ protein is, for example, a drug for regulating various functions of PGC-1 ⁇ protein described above.
- PGC-1 ⁇ protein has various functions. Therefore, adjusting the function of the PGC-1 ⁇ protein is useful for analysis of various biological functions involving the PGC-1 ⁇ protein.
- This PGC-1 ⁇ protein function regulator contains a synoviolin expression inhibitor or a synoviolin activity inhibitor as an active ingredient. Synoviolin expression inhibitors and synoviolin activity inhibitors are known. On the other hand, synoviolin expression inhibitor or synoviolin activity inhibitor can be obtained by obtaining a candidate compound using the screening method described below and examining whether it has synoviolin expression inhibitory activity or synoviolin activity inhibitory activity. You may obtain the inhibitor or the activity inhibitor of Synoviolin.
- the PGC-1 ⁇ protein function regulator of the present invention promotes fatty acid ⁇ oxidation and promotes mitochondrial expression or activity by adjusting the activity of the PGC-1 ⁇ protein. Thus, the function regulator of the PGC-1 ⁇ protein of the present invention is effective for the treatment and prevention of symptoms such as obesity involving the PGC-1 ⁇ protein.
- accession number in the public gene database Genbank of human synoviolin gene is AB024690 (SEQ ID NO: 1).
- the nucleotide sequence of a gene encoding Synoviolin in human is shown in SEQ ID NO: 1. Even proteins other than the protein encoded by the nucleotide sequence have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more). And the protein which has the function which Synoviolin protein has is contained in Synoviolin of this invention.
- “Synoviolin gene” in the present invention includes, for example, an endogenous gene (such as a human synoviolin gene homolog) in other organisms corresponding to the DNA having the nucleotide sequence set forth in SEQ ID NO: 1.
- the endogenous DNA of other organisms corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 1 generally has high homology with the DNA described in SEQ ID NO: 1.
- High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or 96%, 97%, 98% or 99% or more).
- mBLAST algorithm Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7 ) Can be determined.
- stringent conditions for example, “2 ⁇ SSC, 0.1% SDS, 50 ° C.”, “2 ⁇ SSC, 0.1% SDS, 42 ° C.”, “1 ⁇ SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 ⁇ SSC, 0.1% SDS, 65 ° C.”, “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.” and “0.2 ⁇ SSC, 0.1% SDS, 65 ° C.” Can do.
- synoviolin protein (gene)
- the “Synoviolin” of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique.
- the natural protein can be prepared by, for example, a method using affinity chromatography using an antibody against synoviolin protein against an extract of a cell (tissue) considered to express synoviolin protein.
- the recombinant protein can be prepared by culturing cells transformed with DNA encoding synoviolin protein.
- the “Synoviolin protein” of the present invention is suitably used, for example, in the screening method described below.
- “expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of Synoviolin protein” means that transcription and translation of a gene encoding Synoviolin protein occurs or Synoviolin protein is generated by transcription and translation of these genes.
- Synoviolin expression inhibitor or Synoviolin activity inhibitor refers to reducing or eliminating the amount, function or activity of a wild-type Synoviolin gene or protein as compared to the amount, function or activity.
- the “inhibition” includes both inhibition of both function and expression, and inhibition of either one.
- ubiquitination is achieved by repeating a cascade reaction of enzymes such as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), so that the ubiquitin molecule is transferred to the substrate protein.
- enzymes such as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), so that the ubiquitin molecule is transferred to the substrate protein.
- a method for confirming the influence of the test substance on the expression of the synoviolin gene or the influence of the activity of the synoviolin protein for example, a method disclosed in International Publication WO 2006-137514 may be used as appropriate.
- JP 2008-74753 A Patent No. 5008932 discloses plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone) and quercetin (2- (3 , 4-dihydroxyphenyl) -3,5,7-trihydroxy-4H-1-benzopyrano-4-one) is disclosed to inhibit the self-ubiquitination of synoviolin protein.
- Synoviolin self-ubiquitination means ubiquitination of a protein caused by interaction between synoviolins, as disclosed in Japanese Patent Application Laid-Open No. 2008-74753. Protein ubiquitination occurs by synoviolin binding to the protein.
- MBP-Syno ⁇ TM-His means synoviolin deficient in the transmembrane region in which maltose binding protein (MBP) is fused on the N-terminal side and His tag is fused on the C-terminal side.
- Synoviolin expression inhibitor or Synoviolin activity inhibitor examples are plumbagins (2-methyl-5-hydroxy-1,4-naphthoquinone) disclosed in Japanese Patent No. 5008932 And quercetin (2- (3,4-dihydroxyphenyl) -3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a pharmaceutically acceptable salt thereof, or a hydrate thereof.
- synoviolin expression inhibitors or synoviolin activity inhibitors are synoviolin proteins comprising a naphthalene derivative represented by the general formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof It is a ubiquitination activity inhibitor.
- the compound represented by the general formula (I) can be synthesized using a known method.
- Synoviolin protein ubiquitination inhibitor means an agent for inhibiting synoviolin's self-ubiquitination activity. Whether or not to inhibit the ubiquitination activity can be evaluated by measuring the amount of ubiquitinated protein in vitro, for example, as shown in the Examples. As described later, the inhibitor of synoviolin protein ubiquitination activity is effective, for example, as a therapeutic or prophylactic agent for rheumatism and also as a therapeutic or prophylactic agent for obesity.
- the pharmaceutically acceptable salt means a pharmaceutically acceptable salt of the naphthalene derivative represented by the general formula (I).
- the pharmaceutically acceptable solvate means a pharmaceutically acceptable solvate of the naphthalene derivative represented by the general formula (I).
- Examples of pharmaceutically acceptable salts are inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic or basic amino acid salts.
- Examples of inorganic acid salts are hydrochloride, hydrobromide, sulfate, nitrate and phosphate.
- organic acid salts are acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfone Acid salt.
- examples of the inorganic base salt are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, and ammonium salt.
- Examples of organic base salts are diethylamine salt, diethanolamine salt, meglumine salt, and N, N'-dibenzylethylenediamine salt.
- acidic amino acid salts are aspartate and glutamate.
- basic amino acid salts are arginine salts, lysine salts, and ornithine salts.
- An example of a solvate is a hydrate.
- the compound of the present invention can be isolated and purified using known methods by applying ordinary chemical operations such as extraction, concentration, distillation, crystallization, filtration, recrystallization, and various chromatography.
- R 1 to R 4 may be the same or different and each represents a hydrogen atom, a hydroxyl group, a C 1-3 alkyl group, a C 1-3 alkoxy group, or a halogen atom. As demonstrated by the examples described later, at least one of R 1 to R 4 is a hydroxyl group.
- R 5 and R 6 may be the same or different and each represents a hydrogen atom, a C 1-3 alkyl group, or a halogen atom.
- X 1 and X 2 may be the same or different and each represents an oxygen atom or a sulfur atom.
- a 1 -A 2 represents C—C (single bond) or C ⁇ C (double bond). When A 1 -A 2 is C ⁇ C (double bond), formula (I) is represented as formula (II).
- the C 1-3 alkyl group means an alkyl group having 1 to 3 carbon atoms.
- Examples of C 1-3 alkyl groups are methyl, ethyl, n-propyl, and isopropyl.
- a preferred example of the C 1-3 alkyl group is a methyl group.
- C 1-3 alkoxy group means an alkoxy group having 1 to 3 carbon atoms.
- Examples of the C 1-3 alkoxy group are a methoxy group, an ethoxy group, an n-propoxy group, and an isopropoxy group.
- a preferred example of the C 1-3 alkoxy group is a methoxy group.
- halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
- a preferred example of the halogen atom is a chlorine atom.
- a preferred embodiment of the present invention relates to an inhibitor of synoviolin protein ubiquitination activity, wherein at least one of R 1 and R 4 is a hydroxyl group.
- a preferred embodiment of the present invention relates to an inhibitor of synoviolin protein ubiquitination activity in which A 1 -A 2 represents C ⁇ C in the general formula (I).
- This naphthalene derivative is a naphthalene derivative represented by the following general formula (II).
- a preferred embodiment of the present invention relates to a synoviolin protein ubiquitination activity inhibitor in which X 1 and X 2 both represent an oxygen atom and A 1 -A 2 represents C ⁇ C in general formula (I).
- This naphthalene derivative is a naphthoquinone derivative represented by the following general formula (III).
- a preferred embodiment of the present invention is a compound represented by the general formula (I):
- R 1 to R 4 may be the same or different and each represents a hydrogen atom, a hydroxyl group, a methyl group, a methoxy group, or a chlorine atom, wherein at least one of R 1 to R 4 is a hydroxyl group;
- R 5 and R 6 may be the same or different and each represents a hydrogen atom or a methyl group;
- X 1 and X 2 both represent an oxygen atom,
- a preferred embodiment of the present invention is a compound represented by the general formula (I):
- R 1 and R 4 may be the same or different and each represents a hydrogen atom, a hydroxyl group, a methyl group, a methoxy group, or a chlorine atom, wherein at least one of R 1 to R 4 is a hydroxyl group;
- R 2 and R 3 represent a hydrogen atom,
- R 5 and R 6 both represent a hydrogen atom,
- X 1 and X 2 both represent an oxygen atom,
- Preferred embodiments of the present invention include:
- the naphthalene derivative represented by the general formula (I) is 5,8-dihydroxy-4a, 8a-dihydro- [1,4] naphthoquinone, 5-hydroxy-4a, 8a-dihydro- [1,4] naphthoquinone, 5-hydroxy-2,3,4a, 8a-tetrahydro- [1,4] naphthoquinone, 5-hydroxy-7-methoxy-4a, 8a-dihydro- [1,4] naphthoquinone, Any one or more of 5-hydroxy-8-methoxy-4a, 8a-dihydro- [1,4] naphthoquinone and 5-chloro-8-hydroxy-4a, 8a-dihydro- [1,4] naphthoquinone is there, Synoviolin protein ubiquitination inhibitor.
- RNAi is a short double-stranded RNA (hereinafter abbreviated as “dsRNA”) consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence to the cells. This is a phenomenon that induces destruction by specifically and selectively binding to the target gene mRNA, and efficiently inhibiting (suppressing) the expression of the target gene by cleaving the target gene.
- dsRNA short double-stranded RNA
- RNAi when dsRNA is introduced into a cell, expression of a gene having a sequence homologous to that RNA is suppressed (knocked down). Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method using homologous recombination, or a method applicable to gene therapy. It is attracting attention as.
- the RNA used for RNAi is not necessarily completely identical to the Synoviolin gene or a partial region of the gene, but preferably has perfect homology.
- siRNA can be designed as follows.
- (a) There is no particular limitation as long as it is a gene encoding Synoviolin, and any region can be a target candidate.
- an arbitrary region of GenBank accession number AB024690 (SEQ ID NO: 1) can be a candidate.
- (b) A sequence beginning with AA is selected from the selected region, and the length of the sequence is 19 to 25 bases, preferably 19 to 21 bases.
- the GC content of the sequence should be selected, for example, 40-60%.
- Synoviolin siRNA are one base sequence selected from SEQ ID NO: 2 to 6, base sequence complementary to these base sequences, or substitution or insertion of one or two bases from any of these base sequences , RNA having a deleted or added base sequence.
- the RNA having the nucleotide sequences shown in SEQ ID NOs: 2 to 4 is a Synoviolin siRNA that Izumi T, et al., Arthritis Rheum. 2009; 60 (1): 63-72., EMBO, Yamasaki S, et al., EMBO J. 2007; 26 (1): 113-22.
- RNA having the base sequences shown in SEQ ID NOs: 5 and 6 is Synoviolin siRNA, as disclosed in WO 2005/074988 using experiments.
- RNA having a base sequence in which one or two bases have been substituted, inserted, deleted or added from any of these base sequences refers to one base sequence selected from SEQ ID NOs: 2 to 6 and these bases RNA having a base sequence in which one or two bases are substituted, inserted, deleted or added from any base sequence of the base sequence complementary to the sequence. Any one kind of substitution, insertion, deletion or addition may occur, or two or more may occur.
- WO2005-018675 and WO2005 / 074988 disclose siRNAs for genes encoding synoviolin and siRNA screening and evaluation methods for genes encoding synoviolin. Also in the present invention, siRNA against a gene encoding synoviolin can be evaluated by appropriately using the method disclosed in this publication.
- the active ingredient is a base sequence represented by SEQ ID NO: 7 or a base sequence represented by SEQ ID NO: 7, wherein one or two bases are substituted, inserted or deleted Synoviolin decoy nucleic acid having a missing or added nucleotide sequence.
- the nucleic acid having the base sequence represented by SEQ ID NO: 7 is a Synoviolin decoy nucleic acid, for example, using Examples in Tsuchimochi K, et al., Mol Cell Biol. 2005; 25 (16): 7344-56. (SEQ ID NO: 7: 5′-AUGGUGACUGUGUCAUAGA-3 ′).
- Synoviolin decoy nucleic acids and methods for confirming them are known as disclosed in, for example, International Publication WO 2005-093067 and International Publication WO 2005-074988.
- the active ingredient is Synoviolin antisense nucleic acid.
- Methods for screening for Synoviolin antisense nucleic acid and Synoviolin antisense nucleic acid are disclosed in, for example, Japanese Patent Application Laid-Open No. 2009-155204, Table No. 2006-137514, and Table No. 2005-074988.
- Synoviolin antisense nucleic acid means a nucleic acid having a sequence complementary to the synoviolin gene and capable of inhibiting the expression of the synoviolin gene by hybridizing to the gene.
- An antisense nucleic acid can be prepared by a synthetic chemical method or the like by a nucleic acid compound complementary to a partial base sequence of a gene encoding synoviolin.
- a screening test using the gene expression level as an index may be performed.
- said antisense nucleic acid compound for example, the expression of Synoviolin can be suppressed to at least 50% or less as compared with the control.
- antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Genes, Japan Biochemicals) Society, Tokyo Chemistry, 1993, 319-347.).
- the antisense nucleic acid used in the present invention may inhibit the expression and / or function of the synoviolin gene by any of the actions described above.
- an antisense sequence complementary to the untranslated region near the 5 ′ end of the Synoviolin gene mRNA is designed, it is considered effective in inhibiting gene translation.
- a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
- the nucleic acid containing the antisense sequence of the sequence of the untranslated region is included in the antisense nucleic acid used in the present invention.
- the antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
- the nucleic acid thus prepared can be transformed into a desired animal (cell) by using a known method.
- the sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous synoviolin gene or a part thereof possessed by the animal (cell) to be transformed. However, as long as the gene expression can be effectively suppressed, May not be complementary.
- the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene.
- the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases. It is not necessarily limited to this length, and may be, for example, 100 bases or more, or 500 bases or more.
- ribozymes refers to an RNA molecule having catalytic activity. Although ribozymes have various activities, research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Ribozymes include group I introns and M1 contained in RNase P.
- RNA Some have a size of 400 nucleotides or more, such as RNA, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191.).
- the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, and base pairing between U14 and A9 is important for its activity.
- C15, A15 or U15 it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.).
- a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M.
- Hairpin ribozymes are also useful for the purposes of the present invention.
- This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the synoviolin gene transcript in the present invention using a ribozyme.
- RNA interference (hereinafter also referred to as “RNAi”) using double-stranded RNA having the same or similar sequence as the target gene sequence.
- the therapeutic agent of the present invention can be administered either orally or parenterally.
- parenteral administration pulmonary dosage forms (for example, those using a nephriser etc.), nasal dosage forms, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like can be mentioned.
- an injection type it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- the administration method is appropriately selected according to the patient's age and symptoms.
- the effective dose is 0.1 ⁇ g to 100 mg, preferably 1 to 10 ⁇ g per kg body weight at a time.
- the therapeutic agent is not limited to these doses.
- an example of the dosage of the nucleic acid is 0.01 to 10 ⁇ g / ml, preferably 0.1 to 1 ⁇ g / ml.
- the therapeutic agent of the present invention can be formulated according to a conventional method and may contain a pharmaceutically acceptable carrier or additive.
- Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl.
- acceptable surfactants include additives.
- a purified ER stress inducer is dissolved in a solvent (eg, physiological saline, buffer solution, glucose solution, etc.), and Tween 80, Tween 20, gelatin, human serum albumin, etc. are added thereto.
- a solvent eg, physiological saline, buffer solution, glucose solution, etc.
- Tween 80, Tween 20, gelatin, human serum albumin, etc. are added thereto.
- it may be lyophilized to obtain a dosage form that dissolves before use.
- sugar alcohols and saccharides such as mannitol and glucose can be used.
- the second aspect of the present invention relates to a method of screening for a PGC-1 ⁇ protein function regulator. Those that improve the function of the PGC-1 ⁇ protein are preferred. Those that improve the function of PGC-1 ⁇ protein are preferably those that act on anti-obesity.
- a test substance is allowed to act on cells of adipose tissue or individual animals. Thereafter, at least one of the expression level of synoviolin in cells of adipose tissue, the binding between synoviolin and PGC-1 ⁇ protein, and the ubiquitination of PGC-1 ⁇ protein by synoviolin is measured or detected.
- the present invention is based on the finding that inhibiting the activity of Synoviolin leads to an increase in the activity of PGC-1 ⁇ protein.
- Synoviolin a function regulator of PGC-1 ⁇ protein (a substance having an action of regulating the function of PGC-1 ⁇ protein). That is, as a method for screening for a PGC-1 ⁇ protein function regulator, a substance that inhibits the expression and activity of Synoviolin may be screened. Methods for screening for substances that inhibit the expression and function of Synoviolin are described in the present specification and examples, for example, Tokushu 2006/137514, Tokushu 2006/135109, Tokushu 2005/118841. The methods disclosed in Japanese Patent Publication No. 2005, No. 2005/019472, and No. 02/052007 may be used as appropriate.
- An embodiment of the second aspect of the present invention relates to a method for detecting (screening) a mitochondrial activator using the above-described method for screening a function regulator of PGC-1 ⁇ protein.
- An embodiment of the second aspect of the present invention relates to a method for detecting (screening) a therapeutic or prophylactic agent for obesity using the method for screening a function regulator of PGC-1 ⁇ protein described above.
- ⁇ Material Plasmid and antibody> Coding sequences corresponding to full-length mPPAR ⁇ , mPPAR ⁇ , mPGC-1 ⁇ and mPGC-1 ⁇ genes were obtained by PCR amplification from cDNA derived from mouse 3T3-L1 cells. Fragments of PGC-1 ⁇ deletion mutant series were obtained by PCR amplification. The full length PGC-1 ⁇ and each deletion mutant of PGC-1 ⁇ were inserted into pcDNA3 HA vector (modified from Invitrogen) and used for GST pull-down assay and transfection assay. The base sequences of all the plasmids prepared were confirmed by sequence analysis. PPRE ⁇ 3-TK-luc was purchased from addgene Inc.
- Synoviolin conditional knockout mice Synoviolin conditional knockout mice (syno cKO) were prepared by the following method.
- the gene region of 14.8 kb which is the region downstream of exon 16 from the upstream of exon 1 of the mouse synoviolin gene, was used for the construction of the targeting vector.
- a neomycin resistance gene flanked by FRT sequences was inserted between exon 1 and exon 2.
- a loxP sequence was introduced upstream of exon 2 and downstream of exon 14.
- the targeting vector was introduced into ES cells. Clones having the allele in which the desired homologous recombination occurred were selected by confirming the removal of the loxP-exon-loxP sequence by Cre treatment and the removal of FRT-neomycin-FRT by FLP treatment by the length of the PCR product. .
- a clone of the ES cell in which homologous recombination occurred was introduced into a mouse embryo according to a known method (for example, EMBO J 16: 1850-1857) to obtain a chimeric mouse. Furthermore, this chimeric mouse was crossed with a wild type C57BL / 6 mouse to obtain a mouse from which the neomycin sequence was removed. Moreover, since the loxP sequence between the exon and the Long arm incorporated in the targeting vector may be deleted during homologous recombination, its presence was confirmed by PCR.
- mice The obtained neomycin-removed mice were crossed with CAG-Cre mice, and CAG-Cre; syno flox / flox mice (Synoviolin allele and lo-P introduced under control of CMV enhancer and chicken ⁇ -actin promoter and Cre-ER transgene) (Hayashi, S., and McMahon, AP (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation / inactivation in the mouse. Dev Biol 244, 305-318.) In homozygous form. The mice can induce synoviolin knockout with tamoxifen (Tam).
- Tam tamoxifen
- Tamoxifen was administered 7-8 weeks after CAG-Cre; syno flox / flox mice (syno cKO) and homozygous syno flox / flox mice (syno WT) were born.
- the tamoxifen to be administered was dissolved in corn oil (WAKO) to 20 mg / ml. Tamoxifen was administered at a daily dose of 125 mg / kg tamoxifen intraperitoneally for 5 consecutive days. It was confirmed that synoviolin was knocked out by tamoxifen administration by methods such as PCR of genomic synoviolin, real-time PCR of synoviolin mRNA, and Western blotting of synoviolin protein.
- Various primers shown in Table 1 below were used as primers used for real-time PCR.
- Step One Plus (Applied Biosystems) was used as a real-time PCR apparatus.
- Example 2 Observation of mitochondria in Synoviolin knockout mice
- Mitochondria were observed in a conventional manner using an electron microscope in white adipocytes derived from Synoviolin knockout mice (syno cKO) used in Example 1.
- white adipocytes derived from Synoviolin wild type mice (syno WT) were also observed in the same manner.
- the results are shown on the left side of FIG.
- synoviolin knockout mice (syno Adipose) KO) -derived white adipocytes were also observed in the same manner.
- the results are shown on the right side of FIG.
- a fat-specific synoviolin knockout mouse was prepared by first mating a Syvn1 flox / flox mouse and a fatty acid binding protein 4 (aP2) -Cre mouse (Jackson Immunoresearch Laboratories) to aP2-Cre-ER; Syvn1 flox / + mouse. Next, aP2-Cre; Syvn1 flox / + mice were crossed with Syvn1 flox / flox mice as a second mating to obtain aP2-Cre; Syvn1 flox / flox mice. Control mice are mice that have the Syvn1 flox / flox genotype and lack the Cre transgene. 2A and B, the number of mitochondria was remarkably increased in white adipocytes derived from Synoviolin knockout mice, and the size of mitochondria was remarkably increased as compared with white adipocytes derived from Synoviolin wild type mice.
- GST-fused synoviolin lacking the transmembrane domain was incubated with glutathione sepharose 4B.
- the GST fusion protein was incubated with each whole cell extract derived from HEK-293T expressing HA PPAR ⁇ , HA PPAR ⁇ , HA PGC-1 ⁇ or HA PGC-1 ⁇ .
- the bound protein was eluted, separated by SDS-PAGE, and Western blotting using an anti-HA antibody was performed.
- GST not fused with protein was used as a control. The result is shown in FIG.
- Example 4 Binding of Synoviolin and fragmentation mutant PGC-1 ⁇ in vitro
- a GST pull-down assay was performed using a series of PGC-1 ⁇ fragmentation mutations (see FIG. 4). Specifically, a published paper (Aratani, S. st al., (2001). Dual roles of RNA helicase A in (CREB-dependent transcription. Mol Cell Biol 21, 4460-4469.) PGC-1 ⁇ was fragmented by the PCR method. A conceptual diagram of each fragmented PGC-1 ⁇ mutant is shown in FIG.
- FIG. 4 shows that Synoviolin binds to the PGC-1 ⁇ region (195 to 367 amino acid region) containing the LXXLL motif unique to PGC-1 ⁇ .
- the 195 to 367 amino acid region of PGC-1 ⁇ is also a unique sequence that does not exist in PGC-1 ⁇ and PRC.
- Example 5 Binding of fragmented mutant Synoviolin and PGC-1 ⁇ in vitro
- a GST pull-down assay was performed using a series of Synoviolin fragmentation mutations (see FIG. 5 and Yamasaki et al., 2007).
- HA PGC-1 ⁇ prepared by transcription / translation in vitro and Synoviolin fragmented mutant fused to GST or GST were incubated in the same manner as in Example 4, and Western blotting was performed with an anti-HA antibody. The results are shown in FIG.
- FIG. 5 indicates that PGC-1 ⁇ binds to the central part of Synoviolin including a domain unique to and conserved in Synoviolin (236th to 270th amino acid sequence, hereinafter referred to as “SyU domain”). It was also found that only the SyU domain is sufficient for binding to PGC-1 ⁇ , and that a mutant lacking the SyU domain from SynoSyn ⁇ TM (SynoU ⁇ TM ⁇ SyU) cannot bind to PGC-1 ⁇ . From this, it was found that the SyU domain is a minimal domain for binding to PGC-1 ⁇ .
- the protein bound to the anti-FLAG antibody was eluted, separated by SDS-PAGE, and Western blotted with anti-HA antibody and anti-FLAG antibody.
- FIG. 6A From FIG. 6A, HA PGC-1 ⁇ co-immunoprecipitated with SYVN1 / FLAG but not with SYVN1 ⁇ SyU / FLAG. This indicates that synoviolin (SYVN1) is bound to PGC-1 ⁇ via the SyU region in vivo.
- Synoviolin is an ER-resident protein, and PGC-1 ⁇ is known to translocate into the nucleus, so the intracellular localization of synoviolin and PGC-1 ⁇ was examined.
- HEK-293T cells were transfected with HA PGC-1 ⁇ and / or syno / FLAG, synoviolin 3S / FLAG or Synoviolin ⁇ SyU / FLAG expression plasmid, and 24 hours later, synoviolin by immunofluorescence staining using anti-HA antibody and anti-FLAG antibody And the intracellular localization of PGC-1 ⁇ was examined. The results are shown in FIG.
- Synoviolin is known to be an E3 ubiquitinase (Amano, T., et al. (2003). Synoviolin / Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy. Genes Dev 17, 2436 Therefore, it was verified whether PGC-1 ⁇ was a substrate for synoviolin as an E3 ubiquitination enzyme.
- PGC-1 ⁇ FLAG-PGC
- E1-His ubiquitin activating enzyme
- UBE2G2-His ubiquitin-conjugating enzyme
- PK-His-HA-Ub ubiquitin activating enzyme
- FIG. 8A polyubiquitinated PGC-1 ⁇ was detected under conditions where all of ATP, PK-His-HA-Ub, E1-His, UBE2G2-His and Syno (236-338) were present. From this, in It was confirmed that PGC-1 ⁇ is a substrate for Synoviolin in vitro.
- ubiquitination of PGC-1 ⁇ in vivo was examined.
- ubiquitin / FLAG, HA PGC-1, and Synoviolin or Synoviolin 3S expression plasmid were transfected into HEK-293T cells.
- the whole cell extract of HEK-293T cells was immunoprecipitated with anti-HA antibody.
- the bound protein was eluted, separated by SDS-PAGE, and Western blotted with anti-FLAG antibody.
- FIG. 8B From FIG. 8B, HA PGC-1 ⁇ was ubiquitinated in cells expressing Synoviolin WT, but not ubiquitinated in cells expressing Synoviolin 3S.
- MG-132 actually up-regulated the protein content of PGC-1 ⁇ (1.6 times) in dermal fibroblasts treated with solvent (DMSO) as well as tamoxifen-treated cells. In addition, no additive effect was observed between tamoxifen treatment and MG-132 addition (1.1 times).
- siRNA against Synoviolin (Syno) The effect of siRNA was confirmed by the following test.
- Synoviolin knockdown with siRNA was performed in HEK 293 cells.
- SiRNA against Synoviolin has been reported in a published paper (Yamasaki, S., et al. (2007). Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase 'Synoviolin'. See EMBO J 26, 113-122.).
- Transfection of siRNA into cells is performed using Lipofectamine 2000 (Invitrogen) was used according to the protocol. The results are shown in FIG. 9A.
- FIG. 9A confirmed that the synoviolin expression almost completely disappeared in the SynoSyn siRNA-treated cells.
- PGC-1 ⁇ protein level increased 2.5-fold in Syno siRNA-treated cells.
- the expression of PGC-1 ⁇ mRNA was not changed by syno siRNA treatment.
- PGC-1 ⁇ functions as a transcriptional coactivator of several transcription factors such as PPAR ⁇ and PPAR ⁇ , and is known to be involved in various biological events including mitochondrial biogenesis, ⁇ -oxidation and body weight regulation. (Scarpulla, RC (2008). Transcriptional paradigms in mammalian mitochondrial biogenesis and function.Physiol Rev 88, 611-638.) Therefore, PGC-1 ⁇ is syno It was thought to be the causative factor of events observed in cKO mice. To verify this idea, two representative cellular events mediated by PGC-1 ⁇ inhibited in syno cKO were analyzed. One is PGC-1 ⁇ coactivator activity, and the other is mitochondrial biosynthesis.
- HEK-293 cells were treated with control siRNA or Syno.
- siRNA was transiently transfected.
- a reporter plasmid PPRE ⁇ 3-TK-luc
- a CMV- ⁇ -gal expression construct or siRNA was transiently transfected into HEK-293 cells. 16 hours later DMSO
- luciferase assay was performed by treatment with Wy-14643 for 6 hours (FIG. 9B).
- PPAR-luciferase (PPRE ⁇ 3-TK-luc) contains a 3 ⁇ PPAR binding site and is known to be strictly regulated by PPAR, its ligand, and coactivator (Kim, JB, et al. (1998). ADD1 / SREBP1 activates PPARgamma through the production of inherent ligand.Proc Natl Acad Sci U SA 95, 4333-4337.).
- a test was performed in which the amount of Synoviolin expression vector was increased stepwise and simultaneously transfected (FIG. 9C). Sixteen hours after transfection, the cells were treated with solvent (DMSO) or Wy-14643 for 6 hours, and luciferase assay was performed.
- FIG. 9D the number and volume density of mitochondria increased in Syno siRNA-treated cells compared to control siRNA-treated cells, as in the case of syno cKO-derived white adipocytes (FIG. 2).
- Example 10 Inhibition of Synoviolin and PGC-1 ⁇ Binding by Inhibitor of Synoviolin E3 Ubiquitin Ligase Activity
- the inhibitory effect of Synoviolin and PGC-1 ⁇ binding was examined.
- LS-102 PARMACOPIEA
- SDS-PAGE SDS-PAGE with a gel concentration of 12% was added.
- LS-102 is a compound represented by the following structural formula and is a selective inhibitory chemical substance for synoviolin's E3 ubiquitin ligase activity.
- FIG. 10 shows that LS-102 has an inhibitory effect on the binding between Synoviolin and PGC-1 ⁇ .
- Example 11 Screening of a substance that inhibits the binding between Synoviolin and PGC-1 ⁇
- the substances shown in Table 2 below from the screening library related to E3 ubiquitin ligase In as in Examples 3-6 In vitro binding assays were performed. The results are shown in FIGS. 11-1, 11-2, 11-3 and 11-4. Note that 2 ⁇ g of PGC-1 ⁇ B (see FIG. 4) and 2 ⁇ g of GST-Syno ⁇ TM (see FIG. 6) were used as proteins in this example.
- FIG. 11-1 is a Western blot showing the binding inhibitory effect between Synoviolin and PGC-1 ⁇ by the test substances 348 and 349 (upper figure) and a graph evaluating the binding ability of the test substances 348 and 349 (lower figure).
- FIG. 11-2 is a Western blot showing the binding inhibitory effect between Synoviolin and PGC-1 ⁇ by the test substances quercetin and 351 (upper figure), and a graph evaluating the binding ability of the test substances quercetin and 351 (lower figure left and right) It is.
- FIG. 11-3 is a Western blot showing the binding inhibitory effect of each test substance.
- Example 12 Effect of Synoviolin on PGC-1 ⁇ Function of E3 Ubiquitin Ligase Activity Inhibitor and Substance that Inhibits Binding of Synoviolin to PGC-1 ⁇
- Example 9 Example 9 except that 5 ⁇ M LS-102, 0.1 ⁇ M and 0.5 ⁇ M 348, 0.1 ⁇ M and 5 ⁇ M 349, and 5 ⁇ M 351 and 355 were used instead of transfection with Syno siRNA.
- the luciferase assay was performed in the same manner as in 9. As a control, only the same volume of DMSO as a solvent for each compound was added. The results are shown in FIG. From FIG. 12, the luciferase activity decreased only when treated with 351 when treated with the compound.
- Example 13 Effect of a substance that inhibits the binding of Synoviolin and PGC-1 ⁇ on mitochondrial function
- Syno Mitochondria were observed in the same manner as in Example 9 except that 1 ⁇ M 348, 349, 351 and 355 were added to the cells instead of siRNA transfection and observed 72 hours later.
- a photograph is shown in FIG. From FIG. 13, mitochondrial proliferation (increase in number) or increase in mitochondrial size was observed in 348, 349, and 355. From the above, it was suggested that the inhibition of Synoviolin increases the activity of the transcription coactivator PGC-1 ⁇ and activates mitochondria, that is, becomes a completely new molecular target for drug discovery as an agonist.
- 3T3-L1 cells which are mouse fat precursor cells, were cultured for 3 days after reaching confluent in DMEM (Dulbecco's Modified Eagle Medium; High Glucose) containing 10% FBS (fetal bovine serum). Differentiation was induced by adding 500 ⁇ M IBMX (isobutyl-methylxanthine), 1 ⁇ M dexamethasone, and 5 ⁇ g / mL Insulin. Simultaneously, 10 ⁇ M LS-102 (inhibitor of synoviolin ubiquitination activity) or DMSO was added.
- IBMX isobutyl-methylxanthine
- 1 ⁇ M dexamethasone 1 ⁇ M dexamethasone
- siRNA Syno770 sense strand consisting of the sequence of SEQ ID NO: 2 was introduced by Lipofectamine 2000 2 days before differentiation induction.
- the cultured 3T3-L1 cells were washed with PBS ( ⁇ ) (Phosphate Buffered Saline solution excluding magnesium and calcium) and then fixed with 10% formalin. The plate was washed with PBS ( ⁇ ) and replaced with 60% Isopropanol. The cells were stained with 18 mg / mL Oil Red O (solvent: Isopropanol) for 20 minutes, washed with 60% Isopropanol and PBS ( ⁇ ), and observed with a microscope.
- PBS Phosphate Buffered Saline solution excluding magnesium and calcium
- Example 15 Adipocyte Oxygen Consumption in Synoviolin Knockout Mice To examine whether mitochondrial function is activated in CAG-Cre-ER; Syvn1 flox / flox mice, the oxygen consumption of one fat cell was measured.
- FIG. 14 is a graph showing oxygen consumption of adipocytes in Synoviolin knockout mice. Primary mouse adipocytes were isolated and the oxygen consumption of 1 adipocyte was measured. Statistical processing was performed by an independent t-test.
- FIG. 14 shows that the oxygen consumption of fat cells derived from CAG-Cre-ER; Syvn1 flox / flox mice was significantly higher than the oxygen consumption of fat cells derived from control mice.
- Example 16 Basal metabolic rate in Synoviolin knockout mice
- CAG-Cre-ER Basal metabolic rate in Synoviolin knockout mice
- Syvn1 flox / flox mouse To determine whether mitochondrial function is activated, CAG-Cre-ER; was measured basal metabolism of Syvn1 flox / flox mouse (Figure 15).
- FIG. 16 is a photograph replacing a drawing showing Western blotting of adiponectin and synoviolin in each tissue of wild type WT and synoviolin KO.
- alpha-tubulin is an internal standard. From FIG. 16, it is considered that knocking out synoviolin increases the amount of adiponectin and promotes fatty acid combustion.
- Example 18 Measurement of half-life of PGC-1 ⁇
- the half-life of PGC-1 ⁇ was measured. The test is performed by a conventionally known method (Yamasaki, S., et al. EMBO J. 26, 113-122 (2007) and Bernasconi, R., et al. J. Cell Biol. 188, 223-235 (2010)) was modified as follows.
- Mouse embryonic fibroblasts derived from Synoviolin knockout mice (MEF Syno-/-) with 1 ⁇ g pcDNA3 Synoviolin / FLAG, empty vector, or 1.5 ⁇ g pcDNA3 Synoviolin de ⁇ SyU / FLAG, 0.75 ⁇ g pcDNA3 HAPGC-1 ⁇ was transfected. 48 hours after transfection, cells were treated with 40 ⁇ M cycloheximide.
- FIG. 17 shows that wild-type Synoviolin (SYVN1 WT) greatly shortened the half-life of PGC-1 ⁇ , but SYVN1 ⁇ SyU did not significantly promote the degradation of PGC-1 ⁇ . This indicates that the protein level of PGC-1 ⁇ is negatively controlled through binding to Synoviolin in the post-transcriptional process. It was also strongly suggested that Synoviolin is the main E3 ligase of PGC-1 ⁇ .
- Example 19 Effect of Synoviolin Ubiquitination Activity Inhibitor LS-102 on Mitochondrial Function 7 to 8 week-old C57BL / 6J mice were given 50 mg / kg body weight of LS-102 per day or vehicle (DMSO) as a control intraperitoneally, and adipose tissue sections of 57-day mice were observed with an electron microscope. Observed at. The result is shown in FIG. FIG. 18 is an electron micrograph showing the mitochondrial morphology change in adipose tissue by LS-102. The magnification of the electron microscope is 2,500 times and 10,000 times, and the white bars indicate scale bars (2 ⁇ m for 2,500 times and 500 nm for 10,000 times). FIG.
- Example 20 PGC-1 ⁇ half-life MEF (mouse embryoinic) established from Synoviolin KO mice fibriblasts) and an empty vector (control: CONT), synoviolin wild type (SYVN1 WT), and synoviolin unique domain (SYVN1 ⁇ SyU) lacking the binding region with ⁇ according to the standard method.
- CONT synoviolin wild type
- SYVN1 WT synoviolin wild type
- SYVN1 ⁇ SyU synoviolin unique domain
- the cell extract was subjected to Western blotting, and the results are shown in Fig. 19.
- Fig. 19 is a Western blotting showing the half-life of PGC-1 ⁇ , as shown in Fig. 19. The half-life of 4.8 hours, 1.6 hours, and 3.6 hours, respectively. It was.
- the binding assay used the following assay system. 2 ⁇ g MBP-PGC-1 ⁇ His (1-367aa), 2 ⁇ g GST Synoviolin mutant buffered (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% Glucol 1, protease Inhibitor) for 12 hours, and PGC-1 ⁇ was detected with an anti-PGC-1 ⁇ antibody.
- the following assay system was used for the ubiquitin assay. E1-His 125 ng, UbcH5C 150 ng, MBP-SYVN1 ⁇ TM-His 150 ng, GST-PGC-1 ⁇ (1-367 aa; GST-P5), HA-ubiquitin (HA-Ub) 750 ng buffer (50 mM Tris-HCL pH7. (5,5 mM MgCl 2 , 0.6 mM DTT, 2 mM ATP) was reacted at 30 ° C. for 2 hours for ubiquitination.
- FIG. 20 is a diagram showing the results of an in vitro ubiquitination assay.
- FIG. 20 shows that Synoviolin (SYVN1) directly ubiquitinates PGC-1 ⁇ .
- Example 22 Inhibition of ubiquitination of test substance
- SYVN1 Synoviolin
- FIG. 21 is a western blot showing the concentration dependency of the ubiquitination inhibitory activity of PGC-1 ⁇ by the test substances 348 and 349. From FIG. 21, it can be seen that any of the test substances has a higher activity of inhibiting the ubiquitination of PGC-1 ⁇ at 10 ⁇ M than at 1 ⁇ M. That is, the ubiquitination-inhibiting activity of the test substance was shown to be concentration-dependent.
- Example 23 Binding of fragmented mutant Synoviolin and PGC-1 ⁇
- Synoviolin (SYVN1) was shown to have a high possibility of binding to PGC-1 ⁇ in the 236th to 270th region (SyU domain).
- the following experiment was performed in order to find a site having a high possibility of binding to PGC-1 ⁇ in the SyU domain.
- FIG. 22 is the amino acid sequence of the SyU domain in multiple species. SYVN1 SyU mutant in which 5 amino acids corresponding to sites of synoviolin (SYVN1) 236-240, 241-245, 246-250, 251-255, 256-260, 261-265, 266-270 are substituted with alanine, PGC-1 ⁇ was bound.
- FIG. 23 is a Western blot of SYVN1 SyU mutant.
- FIG. 23 shows that the 256-260 and 266-270aa sites are important for binding to PGC-1 ⁇ .
- Example 24 Binding of fragmented mutant Synoviolin and PGC-1 ⁇
- the amino acid sequence at the 266-270aa site is RRAIR.
- Amino acid sequence AAAAA control
- FIG. 24 is a Western blot of mutants of SYVN 1266-270aa.
- FIG. 24 shows that at least two arginine residues are desirable for binding to PGC-1 ⁇ via SYVN 1266-270aa.
- the regulator of mitochondrial function of the present invention can increase the number and size of mitochondria, enhance fatty acid ⁇ oxidation and mitochondrial biogenesis, and can be applied to the treatment or prevention of obesity. Therefore, it can be used in the present invention and the pharmaceutical industry.
- SEQ ID NO: 2 Synthetic RNA Sequence number 3: Synthetic RNA Sequence number 4: Synthetic RNA Sequence number 5: Synthetic RNA Sequence number 6: Synthetic RNA Sequence number 7: Synthetic RNA Sequence number 8: Primer Sequence number 9: Primer Sequence number 10: Primer Sequence number 11: Primer Sequence number 12: Primer Sequence number 13: Primer Sequence number 14: Primer Sequence number 15: Primer Sequence number 16: Primer Sequence number 17: Primer Sequence number 18: Primer Sequence number 19: Primer Sequence number 20: Primer Sequence number 21: Primer Sequence number 22: Primer Sequence number 23: Primer Sequence number 24: Primer Sequence number 25: Primer Sequence number 26: Primer Sequence number 27: Primer Sequence number 28: Primer Sequence number 29: Primer Sequence number 30: Primer Sequence number 31: Primer Sequence number 32: Primer Sequence number 33: Primer Sequence number 34: Primer sequence Issue 35: Primer SEQ ID NO
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Abstract
Description
例えば,特開2008-74753号公報(特許第5008932号)には,プルムバギン(2-メチル-5-ヒドロキシ-1,4-ナフトキノン)及びケルセチン(2-(3,4-ジヒドロキシフェニル)-3,5,7-トリヒドロキシ-4H-1-ベンゾピラノ-4-オン)がシノビオリン蛋白質の自己ユビキチン化を阻害することが開示されている。シノビオリンの自己ユビキチン化とは,特開2008-74753号公報に開示されるように,シノビオリン同士の相互作用により生じるタンパク質のユビキチン化を意味する。タンパク質のユビキチン化は,タンパク質にシノビオリンが結合することにより生じる。
シノビオリンの発現阻害剤又はシノビオリンの活性阻害剤の例は,特許第5008932号に開示されるプルムバギン(2-メチル-5-ヒドロキシ-1,4-ナフトキノン)及びケルセチン(2-(3,4-ジヒドロキシフェニル)-3,5,7-トリヒドロキシ-4H-1-ベンゾピラノ-4-オン),その製薬上許容可能な塩又はこれらの水和物である。
R1~R4は,同一でも異なってもよく,水素原子,水酸基,C1-3アルキル基,C1-3アルコキシ基,及びハロゲン原子のいずれかを表わす。後述する実施例により実証されたとおり,R1~R4の少なくともひとつは水酸基である。
R5及びR6は,同一でも異なってもよく,水素原子,C1-3アルキル基,及びハロゲン原子のいずれかを表わす。
X1及びX2は,同一でも異なってもよく,酸素原子又は硫黄原子を表す。
A1-A2は,C-C(単結合),又はC=C(二重結合)を表す。A1-A2が,C=C(二重結合)の場合,式(I)は,式(II)のように表される。
R1~R4は,同一でも異なってもよく,水素原子,水酸基,メチル基,メトキシ基,又は塩素原子を示し,ここで,R1~R4の少なくともひとつは水酸基であり,
R5及びR6は,同一でも異なってもよく,水素原子,又はメチル基を表し,
X1及びX2は,ともに酸素原子を表し,
A1-A2は,C=Cを表す,
シノビオリン蛋白質のユビキチン化活性阻害剤である。
R1及びR4は,同一でも異なってもよく,水素原子,水酸基,メチル基,メトキシ基,又は塩素原子を表し,ここで,R1~R4の少なくともひとつは水酸基であり,
R2及びR3は,水素原子を表し,
R5及びR6は,ともに水素原子を表し,
X1及びX2は,ともに酸素原子を表し,
A1-A2は,C=Cを表す,
シノビオリン蛋白質のユビキチン化活性阻害剤である。
一般式(I)で示されるナフタレン誘導体が,
5,8-ジヒドロキシ-4a,8a-ジヒドロ-[1,4]ナフトキノン,
5-ヒドロキシ-4a,8a-ジヒドロ-[1,4]ナフトキノン,
5-ヒドロキシ-2,3,4a,8a-テトラヒドロ-[1,4]ナフトキノン,
5-ヒドロキシ-7-メトキシ-4a,8a-ジヒドロ-[1,4]ナフトキノン,
5-ヒドロキシ-8-メトキシ-4a,8a-ジヒドロ-[1,4]ナフトキノン,及び
5-クロロ-8-ヒドロキシ-4a,8a-ジヒドロ-[1,4]ナフトキノンのいずれか又は2つ以上である,
シノビオリン蛋白質のユビキチン化活性阻害剤である。
(a) シノビオリンをコードする遺伝子であれば特に限定されるものではなく,任意の領域を全てターゲット候補とすることが可能である。例えば,ヒトの場合では,GenBank アクセッション番号 AB024690(配列番号:1)の任意の領域を候補にすることができる。
(b) 選択した領域から,AAで始まる配列を選択し,その配列の長さは19~25塩基,好ましくは19~21塩基である。その配列のGC含量は,例えば40~60%となるものを選択するとよい。
(a)シノビオリン遺伝子を発現する細胞に,被験化合物を接触させる工程
(b)前記細胞におけるシノビオリン遺伝子の発現量を測定する工程
(c)被験化合物の非存在下において測定した場合と比較して,発現量を低下させる化合物を選択する工程
配列番号3:5’-GGUGUUCUUUGGGCAACUG-3’
配列番号4:5’-GGUUCUGCUGUACAUGGCC-3’
配列番号5:5’-CGUUCCUGGUACGCCGUCA-3’
配列番号6:5’-GUUUTGGUGACUGGUGCUA-3’
1993, 319-347.)。
RNAのように400ヌクレオチド以上の大きさのものもあるが,ハンマーヘッド型やヘアピン型と呼ばれる40ヌクレオチド程度の活性ドメインを有するものもある(小泉誠および大塚栄子, タンパク質核酸酵素, 1990, 35, 2191.)。
FEBS Lett, 1988, 239, 285.,小泉誠および大塚栄子, タンパク質核酸酵素, 1990, 35, 2191.,Koizumi, M. et al., Nucl
Acids Res, 1989, 17, 7059.)。
323, 349.)。ヘアピン型リボザイムからも,標的特異的なRNA切断リボザイムを作出できることが示されている(Kikuchi, Y.& Sasaki, N., Nucl Acids Res, 1991, 19, 6751.,菊池洋, 化学と生物, 1992, 30, 112.)。このように,リボザイムを用いて本発明におけるシノビオリン遺伝子の転写産物を特異的に切断することで,該遺伝子の発現を阻害することができる。
完全長のmPPARα,mPPARγ,mPGC-1α及びmPGC-1β遺伝子に対応するコード配列を,マウス3T3-L1細胞由来のcDNAからPCRで増幅することによって得た。PGC-1βの欠損変異体シリーズの断片を,PCRで増幅することによって得た。全長のPGC-1βとPGC-1βの各欠損変異体を,pcDNA3 HAベクター(invitrogen社製を改良)に挿入し,GSTプルダウンアッセイやトランスフェクトアッセイに用いた。なお,作製されたすべてのプラスミドの塩基配列は,配列解析によって確認した。PPRE ×3-TK-lucは,addgene Inc.から購入した。シノビオリンプラスミドのシリーズは,既報論文(Amano, T. et al. (2003).
Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for
arthropathy. Genes Dev 17, 2436-2449.及びYamasaki, S. et al. (2007). Cytoplasmic destruction of p53 by
the endoplasmic reticulum-resident ubiquitin ligase 'Synoviolin'. EMBO J 26,
113-122.)のものを用いた。
抗体として,抗FLAG抗体(M2)及び抗tublin抗体はいずれもSigma
Chemical Co製,抗HA-tag抗体(12CA5及び3F10)はRoche製,抗PGC-1β抗体はSant cruze bio製のものを使用した。抗シノビオリンウサギポリクローナル抗体は,既報論文(Yamasaki, S. et al. 2007)に記載されたものを使用した。
シノビオリンコンディショナルノックアウトマウス(syno cKO)を以下の方法で作製した。
Efficient recombination in diverse tissues by a tamoxifen-inducible form of
Cre: a tool for temporally regulated gene activation/inactivation in the mouse.
Dev Biol 244, 305-318.参照)をホモ接合型で有する)を得た。
前記マウスは,シノビオリンノックアウトをタモキシフェン(Tam)によって誘導することができる。
なお,タモキシフェン投与によってシノビオリンがノックアウトされたことは,ゲノム上のシノビオリンをPCRする方法,シノビオリンmRNAをリアルタイムPCRする方法,シノビオリン蛋白質をウエスタンブロッティングする方法などで確認した。
シノビオリンが末梢性のエネルギー消費を変化させる可能性を検討するため,シノビオリンノックアウトマウス由来の白色脂肪細胞においてマイクロアレイを用いた網羅的遺伝子発現解析を行った。
なお,リアルタイムPCRの反応条件は,以下の通りとした。
Stage1(ポリメラーゼ活性化): 95℃ 10min
Stage2(熱変性): 95℃ 1sec
(アニーリング/伸長反応) 60℃ 20sec
Stage3:Stage2を40cycle
RNAを用いて標準化し,syno WTの平均値(n=3)を1として比で表わした。結果を図1に示す。
図1から,β酸化及びミトコンドリア生合成に関与する遺伝子群の転写が増加していることが分かった。したがって,白色脂肪細胞内のミトコンドリアがシノビオリンノックアウトマウスのターゲットとなっていることが示唆された。
実施例1で用いたシノビオリンノックアウトマウス(syno cKO)由来の白色脂肪細胞において電子顕微鏡を用いて常法によりミトコンドリアを観察した。対照としてシノビオリン野生型マウス(syno WT)由来の白色脂肪細胞も同様にして観察した。結果を図2左に示す。
また、脂肪細胞特異的にシノビオリンをノックアウトしたマウス(syno Adipose
KO)由来の白色脂肪細胞についても同様に観察した。結果を図2右に示す。なお、脂肪特異的シノビオリンノックアウトマウスの作製は,まずはSyvn1flox/floxマウスと脂肪酸結合蛋白質4(aP2)-Creマウス(Jackson Immunoresearch Laboratories)とを交配させてaP2-Cre-ER;Syvn1flox/+マウスなどを含む複合ヘテロ接合体を得、次に二次交配としてaP2-Cre;Syvn1flox/+マウスをSyvn1flox/floxマウスと交配させ,aP2-Cre;Syvn1flox/floxマウスを得た。なお,Syvn1flox/floxの遺伝子型を持つ,Creトランスジーンを欠いたマウスを対照マウスとする。
図2A及びBから,シノビオリン野生型マウス由来の白色脂肪細胞に比べ,シノビオリンノックアウトマウス由来の白色脂肪細胞においてミトコンドリアの数が顕著に増加しており,更にミトコンドリアのサイズが顕著に増大していた。
これまで,β酸化とミトコンドリアの複製の両者を制御する活性を有する因子としては脂肪細胞の分化と活性に深くかかわる転写因子ファミリーPPAR(PPARα,PPARγ)とそれに対する転写コアクチベーターPGCファミリー(PGC-1α,PGC-1β,PRC)が知られていた。そこで,前記各因子とシノビオリン蛋白質との結合を確認した。
syno ΔTMは,HA PGC-1βと結合したが,HA PGC-1α,HA PPARγ及びHA PPARαとは結合しなかった。したがって,PGC-1βがシノビオリン蛋白質と選択的に結合することが示された。
シノビオリンと結合するPGC-1βの結合部位を特定するため,PGC-1βの断片化変異のシリーズ(図4参照)を用いてGSTプルダウンアッセイを行った。
具体的には,既報の論文(Aratani, S. st al., (2001). Dual roles of RNA helicase A in
CREB-dependent transcription. Mol Cell Biol 21, 4460-4469.)に準じた方法でPGC-1βの断片化をPCR法にて行った。各断片化PGC-1β変異体の概念図を図4に示す。in vitroで転写/翻訳されたHAタグ付きのPGC-1β断片化変異体とGST又はGST-synoviolin ΔTMとを用いてIn vitro結合アッセイを行った。これらの蛋白質を溶出し,SDS-PAGEで分離し,抗HA抗体でウエスタンブロッティングを行った。結果を図4に示す。
PGC-1βと結合するシノビオリンの領域を特定するため,シノビオリンの断片化変異のシリーズ(図5及び既報論文Yamasaki et al., 2007参照)を用いてGSTプルダウンアッセイを行った。In vitroで転写/翻訳させて作製したHA PGC-1βとGST又はGSTに融合させたシノビオリン断片化変異体とを実施例4と同様にインキュベートし,抗HA抗体でウェスタンブロットを行った。結果を図5に示す。
シノビオリンとPGC-1βとがin vivoで実際に複合体を形成しているか否かを検証した。具体的には,まず,HAタグを付けたPGC-1β(HA
PGC-1β)を発現するプラスミドとFLAGタグを付けたシノビオリン(SYVN1/FLAG)又はシノビオリン変異体(SYVN1ΔSyU/FLAG)を発現するプラスミドとをHEK-293T細胞にトランスフェクトした。前記発現プラスミドをトランスフェクトしたHEK-293T細胞からの全細胞抽出液を準備し,抗FLAG抗体で免疫沈降した。抗FLAG抗体と結合した蛋白質を溶出し,SDS-PAGEで分離し,抗HA抗体及び抗FLAG抗体でウェスタンブロッティングを行った。結果を図6Aに示す。
図6Aから,HA PGC-1βは,SYVN1/FLAGと共免疫沈降したが,SYVN1ΔSyU/FLAGとは共免疫沈降しなかった。このことは,in vivoでシノビオリン(SYVN1)がSyU領域を介してPGC-1βと結合していることを示している。
図6Bから,内在性のPGC-1βは抗シノビオリン抗体と沈殿し,検出された。一方,非免疫マウスIgGでは検出されなかった。このことから,シノビオリンがPGC-1βとin vivoで物理的に結合することが示された。
シノビオリンはER常在性の蛋白質であること,PGC-1βは核内に移行することが知られているため,シノビオリン及びPGC-1βの細胞内局在を調べた。HEK-293T細胞にHA PGC-1β及び/又はsyno/FLAG,synoviolin 3S/FLAG若しくはSynoviolin ΔSyU/FLAG発現プラスミドをトランスフェクトし,24時間後に抗HA抗体及び抗FLAG抗体を用いて免疫蛍光染色によりシノビオリン及びPGC-1βの細胞内局在を調べた。結果を図7に示す。
et al., 2009)されているように,HA PGC-1βは主に核に局在することが分かった。一方,HA
PGC-1βがsyno/FLAGと共発現された場合には,HA PGC-1βは核内ではなく,主に核周辺領域にsyno/FLAGと共局在することが分かった。更に,HA
PGC-1βとSynoviolin ΔSyU/FLAGとを共発現させた場合には,HA PGC-1βは核に局在した。したがって,これらの結果は,シノビオリンがPGC-1βを核周辺領域で捕捉していること,及びin vivoにおけるこの隔離には,SyUドメインが必要であることが示唆された。
シノビオリンは,E3ユビキチン化酵素であることが知られている(Amano, T., et al. (2003). Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy. Genes Dev 17, 2436-2449.参照)ため,PGC-1βがE3ユビキチン化酵素としてのシノビオリンの基質になっていないかを検証した。in vitroで転写及び翻訳させたPGC-1β(FLAG‐PGC),ユビキチン活性化酵素E1(E1-His),ユビキチン結合酵素E2(UBE2G2-His),及びユビキチン(PK-His-HA-Ub)とGST結合シノビオリン変異体(Syno(236-338))とを用いてin vitroユビキチン化アッセイを行った。結果を図8Aに示す。
図8Aから,ATP,PK-His-HA-Ub,E1-His,UBE2G2-His及びSyno(236-338)のすべてが存在する条件下ではポリユビキチン化されたPGC-1βが検出された。このことから、in
vitroにおいてPGC-1βがシノビオリンの基質になっていることが確認された。
図8Bから,シノビオリンWTを発現させた細胞ではHA PGC-1βがユビキチン化されたが,シノビオリン3Sを発現させた細胞ではユビキチン化されなかった。
これらの結果から,PGC-1βはin vitro及びin vivoにおけるシノビオリンの推定上の基質であることが示唆された。
新生後にシノビオリンをノックアウトしたマウス(syno cKO)において,精巣上体及び腸間膜におけるシノビオリン及びPGC-1βの蛋白質レベルをウェスタンブロッティングにより調べた。
図8Cから,シノビオリンノックアウトマウス由来の白色脂肪細胞におけるPGC-1βの蛋白質レベルは,野生型マウス由来のものに比べ,劇的に上昇していた。なお,PGC-1βの転写レベルはシノビオリンノックアウトマウスと野生型マウスとでほとんど差がなかった。
syno CKOマウス由来の皮膚線維芽細胞をDMEM培地で培養し,タモキシフェン(Tam)又は溶媒(DMSO)で48時間処理した。細胞収抽出液又は総RNAを収集し,ウェスタンブロッティング及びリアルタイムPCRをそれぞれ行った。結果を図8Dに示す。
シノビオリンコンディショナルノックアウトマウス由来の皮膚線維芽細胞においてTam処理をした場合PGC-1βの蛋白質レベルは顕著(1.4倍)に上がった。なお,PGC-1βのmRNAレベルは予想通り変化が見られなかった。
これらの結果は,PGC-1βの蛋白質レベルは,転写後のプロセスにおいてシノビオリンによって負に制御されていることを示唆している。また,シノビオリンが細胞内でPGC-1βに対する主なE3であることを強く示唆している。
これまでの遺伝子欠失の効果に加えて,シノビオリンに対するsiRNA(Syno
siRNA)の効果を次の試験により確認した。
まず,HEK 293細胞においてsiRNAによるシノビオリンノックダウンを行った。シノビオリンに対するsiRNAは,既報の論文(Yamasaki, S., et al.
(2007). Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident
ubiquitin ligase 'Synoviolin'. EMBO J 26, 113-122.参照)に従って行った。siRNAの細胞へのトランスフェクトは,Lipofectamine 2000
(Invitrogen社製)を用いてプロトコルに従って行った。結果を図9Aに示す。
paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88,
611-638.参照)。そこで,PGC-1βがsyno
cKOマウスで観察された事象の原因因子であると考えられた。この考えを検証するため,syno cKOにおいて阻害されたPGC-1βを介した2つの代表的な細胞事象を解析した。一つはPGC-1βのコアクチベーター活性であり,もう一つはミトコンドリアの生合成である。
siRNAを一時的にトランスフェクトした。また,PPAR結合部位を含むレポータープラスミド(PPRE ×3-TK-luc),CMV-β-gal発現コンストラクト又はsiRNAをHEK-293細胞に一時的にトランスフェクトした。16時間後 DMSO
または Wy-14643 で 6時間処理しルシフェラーゼアッセイを行った(図9B)。
activates PPARgamma through the production of endogenous ligand. Proc Natl Acad
Sci U S A 95, 4333-4337.参照)。
また,シノビオリンを過剰発現させた効果を調べるため,シノビオリン発現ベクターの量を段階的に増やして同時にトランスフェクトする試験も行った(図9C)。
トランスフェクトした16時間後,細胞を溶媒(DMSO)又はWy-14643で6時間処理し,ルシフェラーゼアッセイを行った。
J., et al. (2003). PGC-1beta in the regulation of hepatic glucose and energy
metabolism. J Biol Chem 278, 30843-30848.参照)の通り,PPARαのアゴニストの一つであるWy-14643は,PPRE ×3-TK-lucレポーター活性を誘導した。Wy-14643誘導条件下において,形質移入されたSyno siRNAは,前記レポーター活性を非常に亢進したが,対照のsiRNAは亢進しなかった。Syno siRNAと同時形質移入されたPGC-1βは,前記レポーター活性を更に亢進した。
図9Cから,シノビオリン過剰発現によってPGC-1βを介したコアクチベーター機能が阻害されることが分かった。
これらの結果から,シノビオリンのノックダウンがPGC-1βに依存した経路を通してPPARαを介した転写を亢進することが示唆された。
図9Dから,syno cKO由来の白色脂肪細胞の場合(図2)と同様に,対照のsiRNA処理細胞に比べてSyno siRNA処理細胞においてミトコンドリアの数及び体積密度が増加した。
シノビオリンのE3ユビキチンリガーゼ活性阻害剤によるPGC-1β機能への影響を調べるため,まずはシノビオリンとPGC-1βとの結合阻害効果を調べた。具体的には,
MBP-PGC―1β-His蛋白質とGST-Syno ΔTM蛋白質とを用いた常法の結合アッセイにおいて,LS-102(PARMACOPIEA社)を添加して12時間結合させ,ゲル濃度12%のSDS-PAGEを行い,次いでウェスタンブロットを行った。一次抗体としては,2500倍希釈した抗GST抗体,二次抗体としては,10000倍希釈した抗ラットHRPを用いた。結果を図10に示す。
なお,LS-102は下記構造式で示される化合物であり,シノビオリンのE3ユビキチンリガーゼ活性に対する選択的阻害化学物質である。
シノビオリンとPGC-1βとの結合を阻害する物質を得るため,E3ユビキチンリガーゼ関連のスクリーニング用ライブラリの中から下記表2に示す物質について,シノビオリンとPGC-1βとの結合への影響を調べるため,実施例3~6と同様のin
vitroでの結合アッセイを行った。結果を図11-1,図11-2,図11-3及び図11-4に示す。なお,本実施例における蛋白質としては,2μgのPGC-1βB(図4参照),及び2μgのGST-Syno ΔTM(図6参照)を用いた。
実施例9において,Syno siRNAをトランスフェクトする代わりに5μMのLS-102,0.1μM及び0.5μMの348,0.1μM及び5μMの349,並びに5μMの351及び355を用いた以外は実施例9と同様にして,ルシフェラーゼアッセイを行った。対照としては,各化合物の溶媒であるDMSOのみを同体積添加した。結果を図12に示す。
図12から,前記化合物で処理した場合において351で処理した場合のみルシフェラーゼ活性が低下した。これらの結果から,シノビオリンとPGC-1βとの結合を阻害することで,PGC-1βの機能を亢進する物質が得られたことが示された。
また,確認としてPGC-1β蛋白質レベルを調べたところ,LS-102処理細胞においてPGC-1βの蛋白質レベルは約2倍に上がった。一方,LS-102処理によってPGC-1βmRNAの発現は変化しなかった。
実施例9において,Syno
siRNAをトランスフェクトする代わりに1μMの348,349,351及び355を細胞に添加し72時間後に観察した以外は,実施例9と同様にしてミトコンドリアを観察した。写真を図13に示す。 図13から,348,349,355においてミトコンドリアの増殖(数の増加)またはミトコンドリアのサイズの増大が観察された。以上から,シノビオリンを抑制することは,転写コアクチベーターPGC-1βの活性をあげ,ミトコンドリアを活性化する,すなわちアゴニストという全く新しい創薬の分子標的となることが示唆された。
マウスの脂肪前駆細胞である3T3-L1細胞を,10%FBS(ウシ胎児血清)含有DMEM(ダルベッコ変法イーグル培地;High Glucose)でconfluentに達した後3日間培養した。500μM IBMX(isobutyl-methylxanthine),1μM Dexamethasone,5 μg/mL Insulinを添加し分化を誘導した。同時に10μM LS-102(シノビオリンのユビキチン化活性阻害剤)もしくはDMSOを添加した。3日間培養後,4μg/mL Insulinを含む培地に置換し10μM LS-102もしくはDMSOを添加した。3日間培養後,10%FBS含有DMEM(High Glucose)に置換し3日間培養した。siRNAに関しては,分化誘導2日前に200pmolのsiRNA Syno770(センス鎖が下記配列番号2の配列からなる)をLipofectamine2000により導入した。
配列番号2:5’-GCUGUGACAGAUGCCAUCA-3’
CAG-Cre-ER;Syvn1flox/flox マウスにおいてミトコンドリアの機能が活性化されているか否かを調べるため,脂肪細胞1細胞の酸素消費量を測定した。
酸素消費量の測定方法は,以下のとおりであった。CAG-Cre-ER;Syvn1flox/flox
マウス及び対照マウスより皮下脂肪を採取し,0.1% (w/v)コラーゲナーゼを用いて37°Cで1時間処理することでシングル細胞化した。得られたシングル細胞の懸濁液について,MitoXpress(登録商標)-Xtra-HS (Luxcel Biosciences Ltd. Ireland)を用い,キットに付属の説明書に従い,酸素消費量を測定した。その結果を図14に示す。図14は,シノビオリンノックアウトマウスにおける脂肪細胞の酸素消費量を示すグラフである。初代マウス脂肪細胞を単離し,脂肪細胞1細胞の酸素消費量を測定した。統計処理は,独立t-検定で行った。
CAG-Cre-ER;Syvn1flox/flox マウスにおいてミトコンドリアの機能が活性化されているか否かを調べるため,CAG-Cre-ER;Syvn1flox/floxマウスの基礎代謝量を測定した(図15)。
基礎代謝量の測定方法は以下のとおりであった。Tam投与後7日目のマウスを用い,4時間絶食させ安静にさせた場合の酸素消費量(VO2)
及び二酸化炭素産生量 (VCO2)を,Oxymax
Equal Flow System (Columbus Instruments, 950 N. Hague Ave; Columbus, OH USA)を用いて測定した。更に,運動活性(運動の数)も一緒にDAS system
(Neuro Science, Inc., Japan)を用いて測定した。図15は,シノビオリンノックアウトマウスにおける基礎代謝量を示すグラフである。統計処理は,独立t-検定で行った。
この結果は, SYVN1が in vivoでミトコンドリア活性を亢進することを示唆している。
野生型WTマウスとシノビオリンKOマウスの各組織におけるアディポネクチンとシノビオリンのウエスタンブロッティングを行った。図16は,野生型WTとシノビオリンKOの各組織におけるアディポネクチンとシノビオリンのウエスタンブロッティングを示す図面に替わる写真である。図中アルファ-チューブリンは,内部標準である。図16から,シノビオリンをノックアウトすることによりアディポネクチンの量が増加し,脂肪酸燃焼が促進されていると考えられる。
SYVN1とPGC-1βとの相互作用がSYVN1を介したPGC-1βの分解に重要であるか否かを確かめるため,PGC-1βの半減期を測定した。
試験は,従来公知の方法(Yamasaki,
S., et al. EMBO J. 26, 113-122 (2007) 及びBernasconi,
R., et al. J. Cell Biol. 188, 223-235 (2010) )を次のように修正して行った。シノビオリンノックアウトマウス由来のマウス胚性線維芽細胞(MEF Syno-/-) に1μg のpcDNA3 Synoviolin/FLAG,空のベクター,又は1.5 μgのpcDNA3 Synoviolin デΔSyU/FLAGと,0.75 μg
pcDNA3 HAPGC-1βとをトランスフェクションした。トランスフェクションから48時間後 ,細胞を40 μM Cycloheximideで
0.5,1,2,又は4時間処理し,細胞をバッファ(10 mM Tris-HCl pH8.0, 150 mM
NaCl, 1mM EDTA, 1 % NP-40, 1 mM DTT, protease inhibitors)で溶解し,抗PGC-1β,抗-SYVN1,又は抗 アルファ-チューブリン抗体で免疫ブロット解析を行った。各試験は少なくとも3回行った。結果を図17に示す。図17から,野生型のシノビオリン(SYVN1 WT)は,PGC-1βの半減期を非常に短縮したが,SYVN1
ΔSyUはPGC-1βの分解をあまり促進していなかった。このことから,PGC-1βの蛋白レベルは転写後のプロセスにおいてシノビオリンとの結合を介した負の制御を受けることが示された。また,シノビオリンがPGC-1βの主要なE3リガーゼであることが強く示唆された。
7~8週齢のC57BL/6Jマウスに,1日当たり50 mg/kg体重のLS-102又は対照としての溶媒(DMSO)を腹腔内に投与し,57日目のマウスの脂肪組織切片を電子顕微鏡で観察した。その結果を図18に示す。図18は,LS-102による脂肪組織のミトコンドリアの形態変化を示す電子顕微鏡写真である。電子顕微鏡の倍率は2,500倍及び10,000倍であり,白抜きのバーはスケールバー(2,500倍の方は2μm,10,000倍の方は500nm)を示す。
図18から,LS-102処理により,脂肪組織細胞におけるミトコンドリアの数と体積密度の増加が起きていることが分かった。このことは,LS-102によりシノビオリンのE3リガーゼ活性が阻害されるとPGC-1βの超活性化が引き起こされミトコンドリアの生合成が促進されることを示唆している。
シノビオリンのKOマウスより樹立したMEF(mouse embryoinic
fibriblasts)にPGC-1βの発現ベクターとともに空ベクター(コントロール:CONT),シノビオリン野生型(SYVN1 WT),シノビオリンユニークドメイン(βとの結合領域を欠失した変異型シノビオリン(SYVN1 ΔSyU)をそれぞれ定法に従い,トランスフェクションした。48時間経過した後に,代表的なタンパク質翻訳阻害剤であるサイクロヘキサミド(40μM)を図に示す時間(0.5時間,1時間,2時間及び4時間)処理し,各細胞抽出液についてウェスタンブロッテングを行った。その結果を図19に示す。図19は,PGC-1βの半減期を示すウエスタンブロッテングである。図19に示されるように,PGC-1βの細胞内での半減期がそれぞれ4.8時間,1.6時間,3.6時間となることが証明された。
結合アッセイは,以下のアッセイ系を用いた。2μgMBP-PGC-1β His(1-367aa),2μgGSTシノビオリン変異体をバッファ(20 mM Tris-HCl pH8.0,100 mM NaCl、1 mM EDTA、0.1 % NP-40、5% グルコールl、プロテアーゼ阻害剤) 内で 12 時間結合させ,抗PGC-1β抗体でPGC-1βを検出した。
被検物質348,349による,シノビオリン(SYVN1)によるPGC-1βのユビキチン化阻害活性の濃度依存性を検討するため,実施例11と同様のインビトロでの結合アッセイを行った。図21は,被検物質348,349によるPGC-1βのユビキチン化阻害活性の濃度依存性を示すウェスタンブロットである。図21から,いずれの被検物質も1μMに比べて10μMにおいてPGC-1βのユビキチン化阻害活性が高いことがわかる。すなわち,被検物質のユビキチン化抑制活性は,濃度依存性があることが示された。
実施例5においてシノビオリン(SYVN1)は236~270番目の領域(SyUドメイン)においてPGC-1βと結合する可能性が高いことが示された。本実施例では,SyUドメインのうちPGC-1βと結合する可能性が高い部位を見出すため,以下の実験を行った。
図22は,複数の種におけるSyUドメインのアミノ酸配列である。シノビオリン(SYVN1)の236-240,241-245,246-250,251-255,256-260,261-265,266-270の部位に相当する5アミノ酸ずつをアラニンに置換したSYVN1 SyU変異体とPGC-1βの結合をおこなった。その結果を図23に示す。図23は,SYVN1 SyU変異体のウェスタンブロットである。図23から,256-260,266-270aaの部位が,PGC-1βとの結合に重要であることが示された。
266-270aaの部位のアミノ酸配列は,RRAIRである。アミノ酸配列がAAAAAであるもの(対照),266番目のRをAに置換したもの(R266A),267番目のRをAに置換したもの(R267A),270番目のRをAに置換したもの(R270A),266番目及び267のRをAに置換したもの(R266,267A),266番目及び270番目のRをAに置換したもの(R266,270A),267番目及び270のRをAに置換したもの(R267,270A),3つのRを全てAに置換したもの(3A)からなるペプチドを用意し,実施例5と同様にしてウェスタンブロットを行った。その結果を図24に示す。図24は,SYVN1266-270aaの変異体のウェスタンブロットである。図24からSYVN1266-270aaを介したPGC-1βとの結合には,少なくとも2つのアルギニン残基が望ましいことがわかる。
配列番号3:合成RNA
配列番号4:合成RNA
配列番号5:合成RNA
配列番号6:合成RNA
配列番号7:合成RNA
配列番号8:プライマー
配列番号9:プライマー
配列番号10:プライマー
配列番号11:プライマー
配列番号12:プライマー
配列番号13:プライマー
配列番号14:プライマー
配列番号15:プライマー
配列番号16:プライマー
配列番号17:プライマー
配列番号18:プライマー
配列番号19:プライマー
配列番号20:プライマー
配列番号21:プライマー
配列番号22:プライマー
配列番号23:プライマー
配列番号24:プライマー
配列番号25:プライマー
配列番号26:プライマー
配列番号27:プライマー
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配列番号30:プライマー
配列番号31:プライマー
配列番号32:プライマー
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配列番号34:プライマー
配列番号35:プライマー
配列番号36:プライマー
配列番号37:プライマー
配列番号38:プライマー
配列番号39:プライマー
配列番号40:プライマー
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配列番号43:プライマー
配列番号44:プライマー
Claims (7)
- シノビオリンの発現阻害剤又はシノビオリンの活性阻害剤を有効成分として含有するPGC-1β蛋白質の機能を調整するPGC-1β蛋白質の機能調整剤。
- 請求項1に記載のPGC-1β蛋白質の機能調整剤であって,
PGC-1β蛋白質による脂肪酸β酸化の促進,及びミトコンドリアの発現又は活性促進のいずれか又は両方のために用いられる,
PGC-1β蛋白質の機能調整剤。 - 請求項1に記載のPGC-1β蛋白質の機能調整剤を含む,
ミトコンドリアの活性化剤。 - 請求項3に記載のミトコンドリアの活性化剤であって,
ミトコンドリアの発現数の増加及びミトコンドリアのサイズの増大のいずれか又は両方を惹き起こすために用いられる,
ミトコンドリアの活性化剤。 - 脂肪組織の細胞又は動物個体に被験物質を作用させ,脂肪組織の細胞における
シノビオリンの発現量,
シノビオリンとPGC-1β蛋白質との結合,及び
シノビオリンによるPGC-1β蛋白質のユビキチン化,
の少なくともいずれかを測定又は検出する工程を含む,
PGC-1β蛋白質の機能調整剤をスクリーニングする方法。 - 請求項5に記載のスクリーニング方法を用いた,ミトコンドリアの活性化剤の検出方法。
- 請求項5に記載のスクリーニング方法を用いた,肥満症の治療剤又は予防剤の検出方法。
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EP2940132A4 (en) * | 2012-12-26 | 2016-08-17 | Nakajima Toshihiro | SCREENING METHOD FOR A CONNECTION WITH EFFECT TO PREVENT OR TREAT ADIPOSITAS |
JP2019156798A (ja) * | 2018-03-16 | 2019-09-19 | 株式会社 バイオミメティクスシンパシーズ | 多発性骨髄腫の治療剤 |
WO2020138219A1 (ja) * | 2018-12-27 | 2020-07-02 | 株式会社nana | 哺乳動物の寿命を延長するための剤 |
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- 2013-12-26 JP JP2014554561A patent/JP6208689B2/ja not_active Expired - Fee Related
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2940132A4 (en) * | 2012-12-26 | 2016-08-17 | Nakajima Toshihiro | SCREENING METHOD FOR A CONNECTION WITH EFFECT TO PREVENT OR TREAT ADIPOSITAS |
JP2019156798A (ja) * | 2018-03-16 | 2019-09-19 | 株式会社 バイオミメティクスシンパシーズ | 多発性骨髄腫の治療剤 |
WO2020138219A1 (ja) * | 2018-12-27 | 2020-07-02 | 株式会社nana | 哺乳動物の寿命を延長するための剤 |
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JPWO2014104224A1 (ja) | 2017-01-19 |
CN105007943A (zh) | 2015-10-28 |
EP2954905A1 (en) | 2015-12-16 |
EP2954905A4 (en) | 2016-11-09 |
EP2954905B1 (en) | 2018-05-02 |
US20150330981A1 (en) | 2015-11-19 |
US9766241B2 (en) | 2017-09-19 |
CN105007943B (zh) | 2017-07-14 |
JP6208689B2 (ja) | 2017-10-04 |
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