JP2020105134A - Pharmaceutical composition for use in treating moyamoya disease or for use in promoting fat metabolism - Google Patents

Abstract

To provide pharmaceutical compositions for use in treating moyamoya disease or for use in promoting fat metabolism, which contain mysterin inhibitor.SOLUTION: Provided is a pharmaceutical composition in which mysterin inhibitor is a mysterin expression inhibitor, and the mysterin expression inhibitor is selected from the group consisting of siRNA, shRNA or expression vectors thereof, antisense oligonucleotide, and miRNA. Also, provided is use as a model of abnormal fat metabolism in a non-human mammal, which has reduced protein expression and/or function of mysterin, or as an individual disorder model due to abnormal aggregate formation of mysterin.SELECTED DRAWING: None

Description

The present invention relates to a pharmaceutical composition for use in treating Moyamoya disease or for promoting fat metabolism.

Moyamoya disease (MMD) is a rare cerebrovascular disorder common in East Asians (Scott and Smith, 2009). MMD is a disease characterized by both left and right stenosis or occlusion of the internal carotid artery in the skull, which often causes cerebral infarction, collateral vessels and life-threatening bleeding. From genetic studies, East Asian of 0.5% to 2% is Misuterin gene; missense mutation in the (m o y amoya ste no- occlusive disease-associated AAA + and R ING finger prote in , also known as RNF213) (R4810K or R4859K) ((Liu et al., 2011; Kamada et al., 2011; Morito et al., 2014; Koizumi et al., 2016)) Is believed to be responsible for the susceptibility to MMD (about 10 times more sensitive to whites).

The mysterin gene has a molecular weight (major isoform) of 591 kDa and has two AAA+ modules and one RING finger ubiquitin ligase domain (see Figure 1A; Liu et al., 2011; Morito et al., 2014). ). AAA+ proteins commonly form a donut-type complex (often a hexamer), generate mechanical forces through structural changes associated with the ATP binding/hydrolysis cycle, and perform biophysical processes. (Eg, so that dynein exerts motor activity) (Ogura and Wilkinson, 2001). Mysterin forms donut-shaped oligomers and has the ability to degrade ATP (Liu et al., 2011; Morito et al., 2014), but what mechanical process mysterin mediates in cells Is unknown. In addition, ubiquitin ligase covalently modifies a substrate protein with a low molecular weight protein, ubiquitin, and performs its proteolytic or functional control (Metzger et al., 2014). Previous studies have suggested that mysterin exerts ubiquitination activity on various substrate proteins including self (Kotani et al., 2017; Liu et al., 2011; Banh et al., 2016). ; Scholz et al., 2016). In addition, mysterin proteins show a diffuse distribution in the cytoplasm and some associate with undetermined intracellular structures (Liu et al., 2011; Morito et al., 2014).

Liu, W. et al., 2011, PLoS One. Vol. 6, Issue 7, e22542 Kamada, F. et al., 2011, J. Hum. Genet. 56:34-40 Morito, D. et al., 2014, Sci. Rep. Vol. 4, Article Number: 4442 Koizumi, A. et al., 2016, Environ. Health Prev. Med. 21:55-70 Ogura, T., and A.J. Wilkinson, 2001, Genes to Cells, 6:575-597. Metzger, M.B. et al., 2014, Biochim. Biophys. Acta-Mol. Cell Res. 1843:47-60. Kotani, Y. et al., 2015, Sci. Rep. Vol. 5, Article Number: 16161 Banh, R.S. et al., 2016, Nat. Cell Biol. 18(7): 803-813 Scholz, B. et al., 201, Dev. Cell. 36:79-93.

The present invention provides a pharmaceutical composition for use in treating Moyamoya disease or for promoting fat metabolism.

The inventors have shown that mysterin wraps lipid droplets and stabilizes them by removing fatty triglyceride lipase (ATGL) from the lipid droplets. The present inventors have revealed that by inhibiting or suppressing the expression of mysterin, the decomposition (or metabolism) of lipid droplets can be promoted and the lipid droplets can be reduced. The present inventors have found a relationship between localization of mysterin in lipid droplets and MMD. The present invention is based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A pharmaceutical composition, which comprises an inhibitor of mysterin, for use in treating Moyamoya disease or for promoting fat metabolism.
(2) The pharmaceutical composition according to (1) above, wherein the mysterin inhibitor is a mysterin activity inhibitor and/or expression inhibitor.
(3) The pharmaceutical composition according to (2) above, wherein the mysterin inhibitor is a mysterin activity inhibitor.
(4) The pharmaceutical composition according to (3) above, wherein the mysterin inhibitor is an AAA+ATPase inhibitor.
(5) The pharmaceutical composition according to (3) above, wherein the mysterin inhibitor is a ubiquitin ligase inhibitor.
(6) The pharmaceutical composition according to (2) above, wherein the mysterin inhibitor is a mysterin expression inhibitor.
(7) The pharmaceutical composition according to (6) above, wherein the expression inhibitor is selected from the group consisting of siRNA against mysterin, shRNA or an expression vector thereof, an antisense oligonucleotide, and miRNA.
(8) Use as a model of abnormal fat metabolism in non-human mammals in which protein expression and/or function of mysterin is reduced, or as an individual disorder model due to abnormal aggregate formation of mysterin.
(9) A method for screening a compound that regulates fat metabolism or a compound that eliminates abnormal myelin aggregation formation,
A method comprising contacting a test compound with mysterin or a variant thereof.

FIG. 1 shows the structure and subcellular localization of mysterin. FIG. 1A shows the basic structure of human mysterin consisting of 5,207 amino acids, the major isoform of mysterin. Mysterin has two adjacent AAA+ modules and one RING finger ubiquitin ligase domain. R4810K (equivalent to the minor isoform R4859K) is a typical missense mutation associated with East Asian Moyamoya disease (MMD). FIG. 1B shows that when mysterin having mCherry at the N-terminus (mCherry-mst) is transiently expressed, a spherical structure having a diameter of about 1 μm is formed in HeLa cells. The rest of the mysterin exhibits a cytosolic distribution. Nuclear chromosomes were stained by Hoechst 33342. Inset is a magnified image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 1C shows that the triglyceride (TG) synthesized on the ER membrane is stored on the hydrophobic surface of the bilayer of the ER membrane and forms spherical lipid droplets (LD) on the cytoplasmic side. LD involves a monolayer of membranes surrounding it and various surface proteins. The right panel shows neutral fat (top row), endogenous PLIN3 (second row from the top), and endogenous ATGL in HeLa cells supplemented with the fatty acid oleic acid (OA). Oleic acid (OA) was used below in the examples to promote LD formation. The scale bar shows 1 μm. FIG. 1D shows that mCherry-mst surrounds neutral fat stained with BODPY 493/503 in HeLa cells. Some LDs were not encapsulated by mysterin (white arrow). It is believed that mCherry-mst can associate transiently with LD or with a particular subset of LD. Inset is a magnified image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 1E shows that endogenous mysterin stained with anti-human mysterin antibody (1C9) is localized in LD. In HeLa cells, treatment with OA promotes LD formation, and treatment with interferon v enhances expression of endogenous mysterin. Inset is a magnified image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 1F shows endogenous mysterin in HepG2 cells stained as described above. Inset is a magnified image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. In FIG. 1G, it is shown that mCherry-mst showed a distributed distribution in HeLa cells in which LD was destroyed by treating with triaxin C, which is a lipid synthesis inhibitor, for 6 hours (left panel). When triaxin C was removed and OA was added, the distribution of LD and mysterin in LD was restored (middle panel). Restoration of LD localization of mysterin was not affected by the addition of Brefeldin A (BFA) (right panel). Inset is a magnified image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 1H shows the recovery of fluorescence after photobleaching. mCherry-mst, mCherry-AUP1, PLIN3-GFP, and GFP-ATGL were overexpressed in HeLa cells to which OA was added (n=5, 5, 7, and 8 respectively). The fluorescence signal showing a donut pattern was quenched by laser irradiation, and the fluorescence recovery (FRAP) was measured. Data are presented as mean ± SEM. From the above, while mysterin shows localized distribution in the cytosol, it is specifically localized in lipid droplets (LD) that are intracellular fat storage organelles, and mysterin is a different lipid droplet protein (AUP1, PLIN3, ATGL) was localized to lipid droplets at a faster rate than that of PLIN3, ATGL), and its localization was independent of COPI (not affected by BFA). FIG. 2 shows that mysterin increases LD by suppressing lipolysis. In FIG. 2A, HeLa cells form LD 48 hours after medium exchange, but LD formation was enhanced when mCherry-mst was overexpressed. The asterisk in the figure indicates cells that do not overexpress mysterin, and o/e indicates cells that overexpress. The scale bar shows 10 μm. In FIG. 2B, the result of having quantified LD in the experiment of FIG. 2A is shown. The number of LDs and the area occupied by LDs were greater in cells overexpressing mCherry-mst (n=32) than in control cells (n=37). Data are presented as mean ± SEM. *** indicates that the p value in comparison with the control is <0.001 and there is a statistically significant difference. FIG. 2C shows that mysterin knockout (KO strains: #1 and #2) using the CRISPR-Cas9 system in HeLa cells leads to lower LD formation than control cells (WT). In these cells, LD formation was previously enhanced by the addition of OA. The scale bar shows 20 μm. FIG. 2D shows the results of quantifying LD in KO cells in the experiment of FIG. 2C. The number of LDs and the area occupied by LDs were significantly reduced in mysterin KO cells (41 cells in #1 strain, 35 cells in #2 strain) compared to control cells (WT, 39 cells). FIG. 2E shows that treatment with triaxin C for 6 hours eliminated LD from HeLa cells under OA-added environment, but overexpression of mCherry-mst was able to maintain LD under the same conditions (upper panel). reference). In FIG. 2E, an asterisk indicates a cell that does not overexpress mysterin, and o/e indicates a cell that overexpresses it. The upper panel shows the results under the presence of OA and triaxin C, and the lower panel shows the results under the presence of OA. FIG. 2F shows the results of quantifying LD in the experiment of FIG. 2E. The number of LD and the area shown by LD were increased in mCherry-mst overexpressing cells (n=29) compared to control cells (n=17). Data are expressed as mean ± SEM. *** indicates a p-value of <0.001 in comparison to the control. FIG. 2G shows that the lipase inhibitor orlistat counteracts the effects of mysterin KO. The upper panel of FIG. 2G is an image of mysterin KO cells to which OA was added, and the lower panel is an image of mysterin KO cells to which orlistat and OA were added. The scale bar shows 20 μm. FIG. 2H shows the result of quantifying LD in the experiment of FIG. 2G. The data obtained from cells not treated with orlistat are the same as in Figure 2D. The number of orlistat-treated cells analyzed was 45 for control cells (WT), 44 for #1 strain, and 40 for #2 strain. Data are expressed as mean ± SEM. ** and *** indicate p-values <0.01 and <0.001, respectively, in comparison with the control cells treated with orlistat, indicating that both have statistically significant differences. From the above, it was shown that mysterin has a function of increasing the number and amount of lipid droplets, and that the function is not the enhancement of the fat synthesis pathway but the inhibition of the lipolysis pathway. Figure 3 shows that mysterin specifically removes ATGL from LD, thereby stabilizing lipid droplets. FIG. 3A shows that the accumulation of ATGL in LD is significantly reduced in mCherry-mst overexpressing cells. LD formation in HeLa cells was stabilized by the addition of OA, whereas cells overexpressing mCherry-mst (indicated by o/e) were compared to control cells not overexpressing mysterin (indicated by an asterisk). , The accumulation of ATGL in LD was significantly reduced. The inset is an enlarged image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 3B shows that mysterin KO in HeLa cells decreases LD (see upper panel) and increases accumulation of endogenous ATGL in LD (see lower panel). LD formation in this experiment is enhanced by the addition of OA. The scale bar shows 20 μm. FIG. 3C is data quantifying the results of the experiment of FIG. 3B. The area represented by ATGL spots was measured and the ratio to total cells is shown as mean ± SEM. *** indicates a p-value of <0.001 in comparison with control (WT). The number of cells analyzed was 20 for WT, 20 for #1 strain and 15 for #2 strain. FIGS. 3D and E show immunostaining images of PLIN3 and ATGL in HeLa cells overexpressing mCherry-mst (by anti-PLIN3 antibody and anti-ATGL antibody, respectively). LD formation is enhanced by the addition of OA. Inset is an enlarged image. The scale bar is 10 μm in the original drawing and 1 μm in the inset drawing. FIG. 3F shows that ATGL siRNA knockdown (KD) significantly increases the reduced LD in mysterin KO. In FIG. 3F, mysterin KO reduces LD formation even in the presence of OA (upper panel), and knockdown of ATGL increases LD formation to the level of target cells (WT). .. From the above, it was shown that the LD enhancing effect of mysterin is mediated by the elimination of ATGL. FIG. 4 shows the observation results of LD formation in various mutants of mysterin and accumulation of the mysterin in LD. FIG. 4A shows the major structure of the enzymatic variant used in FIG. Each mutant has mCherry at its N-terminus. The mysterin A1A2 mutant (hereinafter referred to as "A1A2") is a mutant in which one ATP-binding domain of each of the two AAA+ modules has been introduced to eliminate the ATPase activity. The mysterin B1B2 mutant (hereinafter referred to as “B1B2”) is a mutant in which one amino acid substitution is introduced into each magnesium ion-binding motif essential for hydrolysis of ATP of two AAA+ modules, and each ATPase activity is lost. is there. The RING finger domain-deficient mutant of mysterin (hereinafter referred to as “ΔRING”) is a mutant in which the RING finger domain is completely deleted. FIG. 4B shows that in MyLa cells of mysterin KO (#1), a clear LD formation was observed under the condition of adding OA, whereas a mysterin mutant (A1A2) having two mutations in the ATP binding domain was observed. , A mutant of the magnesium ion-binding motif essential for ATP hydrolysis (B1B2) is not localized in these LDs and has a distributed distribution. Inset is a magnified image. Scale bars are 10 μm and 1 μm in the original and magnified images, respectively. FIG. 4C shows that the mutant lacking the RING finger domain (ΔRING) showed an abnormal aggregate-like pattern in most cells (˜80%) (see left panel) with a small number of cells (˜20%). It shows that LD localization is observed only in (see right panel). Cells (KO #1 HeLa cells) do not express endogenous mysterin and are OA treated. Inset is an enlarged image. Scale bars are 10 μm and 1 μm in the original and magnified images, respectively. FIG. 4D shows the number of LD in cells expressing various mysterin mutants and the area (relative area) occupied by LD in the cells after OA treatment and subsequent triaxin C treatment. Mysterin KO #1 HeLa cells were treated with OA for 16 hours to saturate LD formation. Then, OA was removed, and the obtained cells were treated with triaxin C for 6 hours to stop lipid synthesis. The number of LDs and the relative area were measured. LD was maintained in a small number of cells (ΔR (LD)) that showed localization of mCherry-ΔRING to LD, but LD formation-enhancing effect was observed in cells showing an aggregate-like pattern (ΔR (agg)). It was lost. The number of cells measured was 30 for control cells, 30 for wild-type mysterin-expressing cells (WT), 30 for A1A2-expressing cells, 30 for B1B2-expressing cells, 16 for ΔR (LD) cells, 16 for ΔR (agg) cells. It was 30. All measured data are expressed as mean±SEM. N.S. indicates not significant compared to WT, *** indicates P <0.001. FIG. 4E is a schematic showing that mysterin has an HC subtype of the RING finger domain that chelates two zinc ions with eight conserved cysteine (C)/histidine (H) residues. Four of these are mutated in mutations rarely seen in whites (C3997Y, H4014N, C4017S, and C4032R). FIG. 4F shows that C3997Y (CY) mutant, H4014N (HN) mutant, C4017S (CS) mutant, and C4032R (CR) mutant having a FLAG tag at the C terminus coagulate in many cells (-80%). Although showing an aggregate-like pattern (upper panel), minority of cells (~20%) maintained some LD localization (lower panel). In the experiment, cells that did not express endogenous mysterin (KO #1 HeLa cells) were used for OA treatment. Inset is an enlarged image. Scale bars are 10 μm and 1 μm in the original and magnified images, respectively. FIG. 4G shows the effect of the variants in the four ring finger domains on LD formation. Mysterin KO #1 HeLa cells were treated with OA for 16 hours to saturate LD formation. Then, OA was removed, and the obtained cells were treated with triaxin C for 6 hours to stop lipid synthesis. The number of LDs and the relative area were measured. Cells showing targeting of each variant (LD) to LD retained LD, whereas cells exhibiting an aggregate-like pattern (agg) lost LD retention. The number of cells measured was 30 for agg and 10 for LD for each mutant. All measured data are expressed as mean±SEM. *** indicates that P <0.001. FIG. 4H shows that catanin and Vps4 respectively complex on their substrates, microtubules, or on the ESCRT-III complex. After dissociating the substrate protein complex by ATP hydrolysis, they dissociate. FIG. 4I shows that, based on the above data, mysterin is complexed on LD upon ATP-binding upon removal of ATGL from LD via a mechanical process during ATP hydrolysis, and then diffuses, due to the similarity to catanine/Vps4. The hypothesis of returning to the monomeric form is shown. FIG. 5 shows that mysterin is LD localized (relevant data in FIG. 1). 5A to 5C show that mCherry-mst surrounds the LD in OA-added HeLa cells, HEK293T cells, and HepG2 cells, respectively. HepG2 cells formed larger LD (~2 μm) than HeLa cells. The inset shows a magnified image. Scale bars are 10 μm and 1 μm in the original and magnified images, respectively. FIG. 5D is a Western blot showing the expression of endogenous mysterin in HeLa cells, HEK293T cells, HepG2 cells and preadipocytes and mature adipocytes. Each cell was cultured in the presence or absence of OA, and the cell lysate was electrophoresed and Western blotted. Detection of mysterin was performed using an anti-mysterin antibody (1D6). The arrow indicates the position corresponding to mysterin (591 kDa). A delay in migration was observed only in mature adipocytes in mysterin electrophoresis (see * mark). Mysterin may have been modified during the process of differentiation into adipocytes. The lower Western blot is an internal control with GAPDH. 5E and 5F show that mysterin is induced by interferon v treatment. In FIG. 5E, HeLa cells and in FIG. 5F, HepG2 cells were tested. In mysterin KO cells, no induction of mysterin was observed. The arrow indicates the position corresponding to mysterin (591 kDa). The lower panel is GAPDH internal control. FIG. 5G is a fluorescence microscope image of the LD fraction obtained by cell fractionation. Neutral lipids were verified by staining with LipiDye. LDs obtained from cells overexpressing EGFP-mst retained the mysterin surrounding them. The rapid LD association/dissociation of mysterin (see FIGS. 1H and 4I) appears to have stopped during the sampling process at 4°. 5H to 5J are HEK293T cells expressing mysterin (mst-3×FLAG) having 3×FLAG tag added with OA at the C-terminus, untreated HEK293T cells, and HepG2 cells, respectively, based on density gradient-based cells. The results of fractionation are shown. T is the total amount, LD is the low molecular weight fraction, C is the cytoplasmic fraction, and ppt is the result of Western blotting of the membrane/cytoskeleton fraction. PLIN3 is an LD fraction marker, calnexin is a PPT fraction marker, and GAPDH is a C fraction marker. FIG. 5K shows the distributed distribution of endogenous ATGL in HeLa cells whose LD was destroyed by triaxin C treatment. Removal of triaxin C and addition of OA restored the distribution of LD and ATGL in LD. In contrast, the targeting of ATGL to LD was inhibited by co-addition of Brefeldin A (BFA), in contrast to mysterin. The scale bar shows 10 μm. FIG. 5L shows the maximum recovery of fluorescence after fluorescence quenching based on the data disclosed in FIG. 1H. The recovery rate reflects the operating rate of mCherry-mst on the LD. Data are expressed as mean ± SEM. FIG. 5M shows a half recovery time based on the data disclosed in FIG. 1H. It is suggested that the shorter the time, the faster the shuttling of the fraction in which mCherry-mst operates. Data are expressed as mean ± SEM. From the above, it was confirmed that mysterin was expressed in various cells including adipocytes and hepatocytes and localized in LD. FIG. 6 shows the results of confirming the efficiency of knockout and knockdown of endogenous mysterin. HeLa cells were transformed with pSpCas9(BB)-2A-Puro (PX459) V2.0 (CRISPR-Cas9 system) targeting human mysterin, and the cells were cloned (#1 and #2). FIG. 6A shows the result of Western blot in which the expression of mysterin was detected using an anti-mysterin antibody (1D6). FIG. 6B shows the results of mysterin knockdown. Two independent siRNAs (#1 and #2) knocked down mysterin in HeLa cells. FIG. 6C shows LD formation in the obtained knockdown cells. LD formation was amplified by the addition of OA. LD formation was reduced in knockdown cells. The scale bar shows 20 μm. FIG. 6D shows the triglyceride density in the cell extract in the mysterin KO strain. HeLa cells of the wild-type strain and mysterin KO strain (strains #1 and #2) were lysed with PBS containing 1% Triton X-100 and subjected to TG colorimetric assay. A standard TG was assayed in parallel as a reference. Data are expressed as mean±SEM (n=3 for each sample). *** indicates P<0.001 compared to wild type. It was confirmed that when mysterin was knocked down, fat accumulation was reduced at the cellular level. FIG. 7-1 shows that mysterin specifically changes the distribution of ATGL. FIG. 7-1A shows that mysterin knockdown does not alter ATGL (indicated by arrowhead) protein expression levels in HeLa cells, independent of OA addition. The lower panel shows the internal control (GAPDH). FIG. 7-1B shows the localization of mCherry-mst and AUP1 in HeLa cells overexpressing mCherry-mst. AUP1 was stained with anti-AUP1 antibody. Inset is an enlarged image. Scale bars in the original and magnified images show 10 μm and 1 μm, respectively. Figures 7-1C and 7-1D show that cells overexpressing mCherry-mst (indicated by o/e) are similar to surrounding non-overexpressing cells in PLIN3 (Figure 7-1C). ) And AUP1 (FIG. 7-1D) show normal speckled LD distribution. That is, mysterin does not affect AUP1 and PLIN3. At this time, LD formation was enhanced by the addition of OA. The scale bar shows 20 μm. Magnified images of overexpressing cells are as shown in Figures 3C and 7-1B. In FIG. 7-1E, HeLa cells overexpressing mCherry-mst and PLIN3-GFP were treated with OA to form LD. PLIN3 was always detected in mysterin-covered LD (100%). However, some LD did not contain mysterin but contained PLIN3. This behavior was quite different from ATGL, which never co-localizes with mysterin (0%). The scale bar shows 10 μm. In FIG. 7-1F, HeLa cells overexpressing mst-3×FLAG and mCherry-AUP1 were treated with OA to form LD. AUP1 was always detected in mysterin-covered LD (100%). This behavior was quite different from ATGL, which never co-localizes with mysterin (0%). The scale bar shows 10 μm. In FIG. 7-1G, wild-type HeLa cells and mysterin KD (#1) HeLa cells were treated with BFA (5 μg/mL) for 0, 4, and 8 hours to inhibit ATGL influx into LD. Endogenous ATGL binding to LD was observed at each time point to monitor ATGL efflux (disappearance) from the LD. At this time, OA at a final concentration of 200 μM was added to the cells together with BFA to maintain the amount of LD. Inset is a magnified image. Scale bars in the original and magnified images show 20 μm and 2 μm, respectively. In FIG. 7-1H, wild-type HeLa cells and mysterin KD(#1) HeLa cells were pretreated with BFA (5 μg/mL) for 12 hours to completely deplete ATGL localized in LD. Recovery (influx) of endogenous ATGL in LD was monitored at 0, 8 and 16 hours after removal of BFA by medium exchange. In the above, cells were constantly maintained with the addition of 200 μM OA. The inset shows a magnified image. The scale bars of the original and magnified images are 20 μm and 2 μm, respectively. FIG. 7-1I shows the results of overexpression of GFP-ATGL in OA-added wild-type HeLa cells (n=8) and mysterin KO (#1) cells (n=5). The fluorescence signal showing a donut-shaped pattern was bleached by intense and short laser irradiation, and fluorescence recovery was measured. Data represent mean ± SD. Recovery curves were measured in wild type and KO cells and showed significant differences. ** indicates P<0.01. FIG. 7-1J extracted the recovery immediately after photobleaching from the data shown in FIG. 7-1I. The half-recovery time and the maximum fluorescence recovery rate were calculated using non-linear curve fit analysis to the circular curve. To rule out the effects of LD movement on the second to minute time scale, the initial recovery was measured over approximately 1.6 seconds immediately after photobleaching, and this value was significantly greater in KO cells. This suggests that the molecular exchange of ATGL in LD is significantly faster in KO cells. The half-recovery time was shorter in KO cells and the maximum fluorescence recovery tended to be larger in KO cells, supporting that ATGL recovery in LD was more effective in KO cells. Data are expressed as mean±SD. * Indicates P<0.05. FIG. 7-2K shows the results of overexpression of PLIN3-GFP in wild-type HeLa cells (n=6) and mysterin KO (#1) cells (n=9) to which OA was added. The fluorescence signal showing a donut-shaped pattern was bleached by intense laser irradiation, and fluorescence recovery was measured. Data are expressed as mean±SEM. Unlike ATGL, the recovery curves for PLIN3-GFP were similar in wild type and KO cells. FIG. 7-2L extracted the recovery immediately after photobleaching from the data shown in FIG. 7-2K. The half recovery time and the maximum fluorescence recovery rate were calculated by non-linear curve fit analysis. Data are shown as mean ± SD. * Indicates p<0.05. Figure 7-2M shows that co-expression of GFP-ATGL and mCherry-mst leads to the removal of mCherry-mst from LD. In many cells (-90%; upper panel group), GFP-ATGL showed co-localization with LD, while mCherry-mst showed a dispersive distribution. In a small number of cells (~10%; lower panel group), mCherry-mst showed a donut type distribution in LD, but did not co-localize with GFP-ATGL, which indicates that mysterin and ATGL had LD. Suggests that they compete with each other for localization to. The nuclear chromosome was stained with Hoechst33342. Inset is an enlarged image. The scale bars in the original and inset images were 20 μm and 2 μm, respectively. Figure 7-2N shows a model of competition in LD between mysterin and ATGL. (A) ATGL is distributed in LD through the ER-LD binding site. It was speculated that mysterin probably prevented the influx of ATGL into the LD at this stage, and ATGL removed by mysterin was retained at the pre-LD site, ER. (B) Overexpression of ATGL reduces the amount of mysterin in LD, suggesting that ATGL prevents myelin influx into LD (or promotes myelin efflux from LD). Without direct interaction between mysterin and ATGL (see Figure 7-20), mysterin and ATGL may compete for an unidentified anchor protein. Figures 7-20 show that mysterin and ATGL do not interact directly. HEK293 cells transiently expressing Mst-3×FLAG were lysed and subjected to immunoprecipitation with anti-FLAG M2 agarose. Cell lysates and co-immunoprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblot with anti-ATGL antibody. No co-immunoprecipitation of mysterin and ATGL was observed. The point that mysterin is detected as a smear-like band pattern may be due to the treatment in electrophoresis, which is as discussed in (Kotani et al., 2017). FIG. 7-2P shows the results of knocking down ATGL with siRNA in HeLa cells of mysterin KO (#1) and HepG2 cells treated with three independent siRNAs (#1 to #3). The expression of ATGL was examined by immunoblotting using an anti-ATGL antibody, and the effect of ATGL knockdown performed in FIG. 3F could be confirmed. The lower panel group shows the internal control (PLIN3). From the above, it was confirmed that mysterin specifically acts on ATGL. FIG. 8 shows the molecular behavior of enzymatic and pathogenic variants. FIG. 8A shows that cells overexpressing mCherry-ΔRING (indicated by o/e) show two patterns. The majority cells (-80%; upper panel) showed aggregation-like structure formation of ΔRING mutants and normal localization of endogenous ATGL to LD. A small number of cells (~20%; lower panel group) had LDs covered by ΔRING mysterin, but ATGL could not localize to LDs surrounded by ΔRING mysterin. This suggests that mysterin ubiquitin ligase activity is not directly required for ATGL clearance. Cells that do not overexpress mysterin (indicated by *) show LD highly covered with ATGL (upper and lower panel groups). The scale bar shows 20 μm. FIG. 8B shows that the D4013N mutant and the R4810 mutant (both labeled with mCherry) were normally LD-localized in myelin KO#1 HeLa cells to which OA was added. Inset is an enlarged image. Scale bars in the original and magnified images are 10 μm and 1 μm, respectively. The position of the mutation in the total length of mysterin is indicated in the upper panel. The aspartic acid residue at position 4013 exists adjacent to the conserved histidine residue at position 4014 (see FIG. 4E), but it is considered not essential for enzymatic activity. R4810K is distributed in the C-terminal region without the putative functional structure. The genetic penetrance of these mutations is incomplete. FIG. 8C shows LD formation in the R4810K mutant (shown as RK) and D4013N mutant (shown as DN). Mysterin KO#1 HeLa cells were treated with OA for 16 hours to saturate LD formation. OA was removed and cells were treated with triaxin C for 6 hours to stop lipid synthesis. The number of LD and the relative area were quantified. Data for mock treated and WT cells are identical to Figures 4D and 4E. The R4810K (RK) mutant and the D4013N (DN) mutant were localized in LD as in the wild type (WT). The number of cells quantified was 26 for RK and 29 for DN. Data are shown as mean ± SEM. N.S. indicates not significant and *** indicates p<0.001 compared to wild type. FIG. 8D shows a model in which the binding of ATP to the first AAA+ module of mysterin induces oligomerization and is dissociated by hydrolysis of another ATP in the second AAA+ module. From the above, it was confirmed that both the AAA+ ATPase activity and the ubiquitin ligase activity of mysterin are important for LD localization and function of mysterin, and this function is impaired by pathogenic mutations found in moyamoya disease patients.

Detailed description of the invention

As used herein, the term “subject” means a mammal, and particularly includes a human.

The term “treatment” is used herein to include therapeutic treatment and prophylactic treatment. As used herein, “treatment” means the treatment, cure, prevention or amelioration of a disease or disorder or the reduction of the rate of progression of the disease or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.

In the present specification, the term “fat metabolism” is used to mean a fat catabolism. Fat metabolism includes cell fat metabolism and organ fat metabolism.

As used herein, “inhibitor” means an agent capable of inhibiting the function of the target protein. Examples of the inhibitor include chemical substances (low molecular weight compounds), peptides, nucleic acids (for example, siRNA, shRNA, and expression vectors thereof, antisense oligonucleotides, miRNA). The inhibitor may preferably have specificity or selectivity for its target protein. Having specificity or selectivity means having an IC ₅₀ value lower than that for any other target protein, preferably less than 50%, less than 40%, less than 30%, less than 20%. It is less than 10%, less than 1/100, less than 1/1000 or less than 1/10000.

As used herein, the “expression inhibitor” means an agent capable of inhibiting the expression of a target protein. Examples of the expression inhibitor include nucleic acids (for example, siRNA, shRNA, and expression vectors thereof, antisense oligonucleotides, miRNA) targeted at mRNA encoding a target protein. Expression of the target protein can also be suppressed by recruiting a Cas9 (dCas9) protein having no endonuclease activity to the promoter region or transcription initiation region of the target protein using gRNA (or crRNA and tracrRNA) .. The mechanism of such expression suppression is called CRISPRi. The method for designing gRNA and crRNA is well known, and about 20 mers at the 5'end of gRNA or crRNA can be designed to be hybridizable to a target sequence under physiological conditions.

As used herein, “mysterin” is a protein containing a ring finger domain and an AAA+ATPase domain, and is a ring finger protein 213 (RFP213), an ALK lymphoma oligomerization partner on chromosome 17 on chromosome 17. ALO17), Moyamoya disease 2 (MYMY2), mysterin (MYSTR), NET57, chromosome 17 open reading frame 27 (C17orf27), or KIAA1618.
Mysterin has a ring finger domain, has the ability to bind up to two zinc ions, ubiquitin ligase activity (activity that links ubiquitin to protein), and two AAA+ ATPase domains. Have activity. Mysterin can ubiquitinate itself and other proteins through the activity of the ring finger domain.
The “mysterin” can be human mysterin, which can have the amino acid sequence registered under UniProtKB as accession number: Q63HN8. The base sequence encoding human mysterin may have the base sequence registered under GenBank accession number: NM_020914.4. For human mysterin, isoform 2 having an amino acid sequence registered as accession number: Q63HN8-4 in UniProtKB is known. There are naturally several variants of mysterin. Mysterin is known to be a causative gene of moyamoya disease.

As used herein, mysterin binds to the intracellular lipid droplet surface and stabilizes the lipid droplet. Part of this fat stabilizing effect is exerted by removing fat triglyceride lipase (ATGL) from fat droplets. Inhibiting either the AAA+ ATPase activity, the ubiquitin ligase activity, or the expression of mysterin reduces lipid droplets. From this, it was found that inhibition of mysterin in cells enhances fat metabolism in cells.

Accordingly, the present invention provides a pharmaceutical composition for use in promoting fat metabolism, which comprises an inhibitor of mysterin.

In addition, mysterin inhibitors can be used to treat diseases associated with the accumulation of lipid droplets. Lipid droplets are observed in patients with diseases such as overweight, obesity and fatty liver. Therefore, inhibitors of mysterin can be used to treat patients with disorders such as obesity and fatty liver. In addition, mysterin inhibitors can be used to treat lifestyle-related diseases caused by the accumulation of lipid droplets. Examples of lifestyle-related diseases caused by accumulation of fat droplets include diseases such as diabetes, non-alcoholic hepatitis, liver cirrhosis, and arteriosclerosis.

Accumulation of lipid droplets is, for example, excessive accumulation of lipid droplets. Excessive accumulation of lipid droplets is, for example, compared to the accumulation of lipid droplets of a healthy person, a specific value between the average value and the maximum value, or a specific value between the average value and the third quartile. It can be evaluated that it is accompanied by accumulation of lipid droplets when it is higher than the value of. Accumulation of lipid droplets is also accompanied by accumulation of lipid droplets when it is higher than a certain value between the mean value and the mean value of +σ, +2σ, or +3σ, as compared with the accumulation of lipid droplets of a healthy person Can be evaluated. Here, σ is the standard deviation of the population of healthy subjects.

Accumulation of lipid droplets can be quantitatively evaluated by observing lipid droplets in cells quantitatively. It can also be investigated by separating lipid droplets from tissues or cells and quantifying triglyceride in the separated lipid droplets. Quantitative observation of fat droplets and triglyceride quantification can be performed by methods well known to those skilled in the art. The quantitative observation of the lipid droplets can be quantified by, for example, fluorescently labeling the triglyceride in the lipid droplets and evaluating the number of lipid droplets and/or the lipid droplet area using a fluorescence microscope. The triglyceride can be quantified by, for example, hydrolyzing the triglyceride with lipase and measuring the obtained free glycerol by HPLC, a light scattering detection technique, or the like. Triglyceride can also be quantified by the sulfo-phospho-vanillin method. In the sulfo-phospho-vanillin method, concentrated sulfuric acid is added to lipid droplets separated from tissues or cells, heated to solubilize the sample, and vanillin is added to measure the colorimetric product, which results in lipid droplets. Can be quantified. Extraction of fat droplets can be performed, for example, using a fat-soluble organic solvent (eg, chloroform) (eg, by the Folch method). The lipid droplets are also quantified by suspending cells or tissues in physiological saline containing a nonionic surfactant and using a commercially available colorimetric quantification kit (eg, Serum Triglyceride Quantification Kit, CELL BIOLABS, INC.). be able to.

According to the present invention, when mysterin expression is eliminated or reduced by knocking out or knocking down mysterin, recruitment of ATGL to lipid droplets is restored, and ATGL decomposition of lipid droplets is promoted. Therefore, the fat droplet becomes smaller. Therefore, according to the present invention, there is provided a pharmaceutical composition containing a mysterin expression inhibitor for use in promoting fat metabolism.

Examples of mysterin expression inhibitors include nucleic acids that target mysterin. Examples of the nucleic acid targeting mysterin include siRNA, shRNA, miRNA, or antisense oligonucleotides targeting mysterin, or intracellular expression vectors thereof. Methods for designing siRNA, shRNA, and antisense oligos are well known to those of skill in the art. Therefore, siRNA, shRNA, and antisense oligo can be designed and used by methods well known to those skilled in the art. Also, these nucleic acids can include cross-linked nucleic acids (BNA), locked nucleic acids (LNA), and modified oligonucleotides such as morpholino oligos. The nucleic acid may include one or more selected from the group consisting of 2'-O-methylated RNA, and 2'-fluoro modified RNA. Nucleic acids may also be linked between bases by phosphorothioate linking groups. In certain aspects, siRNAs and shRNAs can include a nucleic acid having the sequence of SEQ ID NO:2 and a nucleic acid having the sequence of SEQ ID NO:3.

The expression inhibitor of mysterin also includes microRNA. In the present invention, it is not particularly limited as long as it is a microRNA capable of suppressing the expression of mysterin, but for example, let-7c can be used.

The mysterin expression inhibitor also includes a CRISPR-off system targeting mysterin. A mysterin-targeted CRISPRi may comprise a dCas9 protein and a combination of gRNA or crRNA and tracrRNA for recruiting dCAS9 to the promoter or transcription initiation region of mysterin. The dCAS9 protein is a Cas9 protein in which the endonuclease activity of the two endonuclease domains possessed by the Cas9 endonuclease is inactivated. The dCas9 protein recruited to the promoter region or transcription initiation region of mysterin forms a physical obstacle on the transcribed DNA, which leads to the recruitment of RNA polymerase II to the promoter and transcription elongation of mRNA by RNA polymerase II. Can inhibit the transcription of mRNA. In order to inactivate the Cas9 endonuclease, for example, in Cas9 protein of Streptococcus pyogenes, amino acid mutations of D10A and H840A can be introduced. In Cas9 proteins of other species, amino acid mutations in two endonuclease domains corresponding to D10A and H840A can be introduced. The dCas9 protein may further have a nuclear localization signal. Further, the dCas9 protein may be a fusion protein with a transcription repression domain (eg, KRAB; Kruppel-associated Box).

Examples of the mysterin expression inhibitor include a gene expression vector containing a nucleic acid having a promoter operably linked with a gene (nucleic acid) encoding one or more selected from siRNA, shRNA, and antisense oligo. As the promoter, a promoter of RNA polymerase III such as U6 promoter can be used. The mysterin expression inhibitor also includes a nucleic acid encoding a dCas9 protein operably linked to a promoter or a nucleic acid encoding a fusion protein of a dCas9 protein and a transcription repression domain, and a gRNA or crRNA operably linked to a promoter. It may be a gene expression vector containing a combination with tracrRNA. The combination of a nucleic acid encoding a dCas9 protein operably linked to a promoter or a nucleic acid encoding a fusion protein of a dCas9 protein and a transcription repression domain, and a combination of gRNA or crRNA and tracrRNA operably linked to a promoter is 2 or more. The gene expression vector can be contained separately, and preferably can be contained in one gene expression vector.

Mysterin can also inhibit its lipid droplet stabilizing effect by inhibiting its AAA+ ATPase activity. Therefore, according to the present invention, there is provided a pharmaceutical composition for use in promoting fat metabolism, which comprises an inhibitor of myasterin AAA+ ATPase activity. Inhibitors of mysterin AAA+ ATPase activity are those that can bind mysterin, eg, can bind the mysterin AAA+ module. As the inhibitor of AAA+ATPase activity of mysterin, for example, a known inhibitor of AAA+ATPase activity can be used.

Mysterin can also alter its subcellular localization by inhibiting its ubiquitin ligase activity, thereby inhibiting its lipid droplet stabilizing effect. Accordingly, the present invention provides a pharmaceutical composition for use in promoting fat metabolism, which comprises an inhibitor of mysterin ubiquitin ligase activity. Inhibitors of mysterin ubiquitin ligase activity are those capable of binding mysterin, eg, the ring finger domain of mysterin. As the inhibitor of mysterin ubiquitin ligase activity, for example, a known ubiquitin ligase inhibitor can be used.

Any of the above mysterin inhibitors can be used to treat Moyamoya disease. Accordingly, the present invention provides a pharmaceutical composition containing an inhibitor of mysterin for use in treating Moyamoya disease. According to the present invention, there is provided a pharmaceutical composition containing a mysterin expression inhibitor or an inhibitor of AAA+ATPase activity, for use in treating moyamoya disease.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient, carrier and/or diluent in addition to the inhibitor of mysterin. By "medically acceptable" is meant not causing a medically unacceptable adverse reaction when administered to a subject. Typically, such compositions are prepared as injectables, as liquid solutions or suspensions, and solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
Excipients, carriers or diluents include, for example, any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agent, isotonic and absorption delaying agents, buffers, carrier solutions, suspending agents. Suspensions, colloids, etc. are included. Buffers of phosphates, citrates, and other organic acid salts; antioxidants including ascorbic acid; hydrophobic polymers (eg polyvinylpyrrolidone); amino acids (eg glycine, glutamine, asparagine, arginine or lysine); glucose, Monosaccharides, disaccharides and other carbohydrates including mannose or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants (eg, Examples include polyoxyalkylene type). The use of such media and agents for pharmaceutically active substances is well known in the art. Its use in therapeutic compositions is contemplated, unless any conventional vehicle or substance is incompatible with the active ingredient.

Another aspect of the invention provides the use of an inhibitor of mysterin in the manufacture of a pharmaceutical composition for use in treating Moyamoya disease. Another aspect of the invention provides the use of an inhibitor of mysterin in the manufacture of a pharmaceutical composition for use in promoting fat metabolism.

In another aspect of the invention, there is provided a method of treating Moyamoya disease in a subject in need thereof, comprising administering to the subject an inhibitor of mysterin. In another aspect of the invention, there is provided a method of promoting fat metabolism in a subject in need thereof, comprising administering to said subject an inhibitor of mysterin.

In another aspect of the present invention, a non-human mammal having reduced or defective protein expression and/or function of mysterin is provided. Here, non-human mammals include primates (for example, monkeys, chimpanzees, orangutans, gorillas, bonobos), livestock (for example, cows, horses, sheep, goats, pigs), and pets (dogs, cats, mice, squirrels). , Rabbits, hamsters). Mysterin may be an orthologue of human mysterin having an amino acid sequence under accession number: Q63HN8 in UniProtKB in each animal. Also, mysterin may be an ortholog of human mysterin having an amino acid sequence registered under accession number: Q63HN8-4 in UniProtKB. The decrease in the expression of mysterin can be performed by gene knockout or gene knockdown. Gene knockdown can be performed by, for example, a vector expressing an shRNA against mysterin. Gene knockout can be performed due to gene deletion or the like. In addition, a method of stopping gene expression such as CRISPR-OFF using dCas9 or dCas9 and a transcription repression domain may be used. The non-human mammal in which the protein expression and/or function of mysterin is reduced or deficient may be a model of abnormal fat metabolism (eg, model of increased lipid metabolism). Therefore, according to the present invention, there is provided use as a model of abnormal lipid metabolism in a non-human mammal (for example, a model for enhancing lipid metabolism) in which mysterin protein expression and/or function is reduced or deleted. The present invention also provides an individual dysfunction model resulting from cytotoxicity associated with the formation of an aggregate-like structure of mutant mysterin. In a model of individual dysfunction caused by cytotoxicity associated with the formation of an aggregate-like structure of mutant mysterin, the model animal has amino acid mutations corresponding to one or more mutations selected from C3997Y, H4014N, C4017S, and C4032R. obtain.
In another aspect of the present invention, a non-human mammal having improved protein expression and/or function of mysterin is provided. Non-human mammals include those mentioned above. To improve the protein expression of mysterin, a technique such as transgenic can be used. In addition, a method of enhancing gene expression such as CRISPR-ON may be used. It may be a fat metabolism disorder model (eg, lipid metabolism suppression model). Therefore, according to the present invention, there is provided use of mysterin as a model for abnormal lipid metabolism in a non-human mammal (for example, a model for suppressing lipid metabolism) in which protein expression and/or function of mysterin is improved.
Examples of the use of the present invention as the above-mentioned fat metabolism abnormality model and individual disorder model include use of the model for research purposes, use of the model for drug screening purposes, and the like.

In another aspect of the present invention, a method for screening a compound that regulates fat metabolism,
Methods are provided that include contacting a test compound with mysterin or a variant thereof. As the mysterin mutant, a mutant in which the activity of the mysterin in the AAA+ module is lost, a mutant in which the AAA+ module is destroyed, a mutant in which the activity in the ring finger domain is lost, or a mutant in which the ring finger domain is destroyed is used. obtain. A variant of mysterin is also a mysterin having either or both ubiquitin ligase activity and ATPase activity in the AAA+ module, which is 90%, 95%, 96%, 97%, 98% of the amino acid sequence of human mysterin. Mysterins having amino acid sequences with%, 99% or 99.5% or greater homology can be used. A mutant of mysterin is also a mysterin having either or both of a ubiquitin ligase activity and an ATPase activity in the AAA+ module, which has a mutation of 1 to 30 amino acids with respect to the amino acid sequence of human mysterin. And the mutations can independently use variants selected from deletions, additions, insertions and/or substitutions. As the mysterin, an ortholog of a non-human mammal can be used. Modulation is used in the sense of including promotion and/or inhibition.
The contacting step of the screening method of the present invention can be performed in an aqueous solution containing the test compound and mysterin or a mutant thereof. The contacting step of the screening method of the present invention can be performed by contacting the test compound with cells expressing mysterin or a mutant thereof. The contact step of the screening method of the present invention can be performed by administering a test compound to a non-human mammal expressing mysterin or a variant thereof (eg, oral, intravenous, intracerebroventricular, intraperitoneal, etc.). ..
The contacting step of the screening method of the present invention comprises the non-human mammal in which the protein expression and/or function of mysterin of the present invention is reduced or deleted, and the non-human mammal in which the protein expression and/or function of mysterin of the present invention is improved. It may be performed by administering a test compound to a mammal.
The test compound library for screening is not particularly limited, and for example, a natural product library, a low molecular compound library, a compound having an ATPase activity inhibitory effect, a library containing a kinase activity inhibitor, etc. can be used.

In addition, a mysterin expression inhibitor or an AAA+ ATPase activity inhibitor can eliminate abnormal aggregate-like structure formation due to the disease mutant mysterin, and therefore is exerted by forming such an abnormal aggregate-like structure. It can be used to treat diseases caused by cytotoxicity. Examples of the diseases caused by the cytotoxicity exhibited by the disease mutant mysterin forming an aggregate-like structure include moyamoya disease, hypertension, cerebral aneurysm, myocardial infarction and the like.

Materials and experimental methods [cells, transfection, and treatment of cells]
HeLa (Kyoto), HEK293T, and HepG2 cells were treated with Dulbecco's modified Eagle medium (DMEM; Gibco) containing 10% fetal bovine serum (Sigma) and penicillin/streptomycin (final concentration 100 U/mL and 100 μg/mL; Nacalai Tesque, respectively). ) At 37° C. in the presence of 7% CO ₂ . Human visceral preadipocytes (Lonza) were maintained using the PGM-2 Bullet Kit (Lonza) according to the manufacturer's instructions and allowed to differentiate into mature adipocytes.
The plasmid was transfected into cells using Polyethylenimine MAX (Polysciences), and the siRNA was transfected into cells using RNAiMAX (Thermo Fisher Scientific). Oleic acid (OA; Merck) was conjugated to bovine serum albumin (BSA; fatty acids and endotoxin free; Sigma) in phosphate buffered saline (PBS) at a 6:1 molar ratio before use. Cells were treated with OA/BSA complexes containing a final concentration of 200-400 μM OA or corresponding concentrations of BSA alone for 16-24 hours. Triacsin C (Sigma) and orlistat (AdooQ BioScience) were diluted in DMSO and added to the medium at a final concentration of 5 μM for 6-24 hours and a final concentration of 100 μM for 24-48 hours, respectively. Interferon ν (PBL Assay Science) was added for 24 hours at the concentration shown in the figure. Brefeldin A (Nacalai Tesque) was added at a final concentration of 5 μg/mL for 6-24 hours.
[Plasmid and siRNA]
Wild-type and mutants with three tandem FLAG epitopes (mst-3xFLAG) at the C-terminus and EGFP (EGFP-mst) at the N-terminus have been previously described ((Liu et al., 2011. Morito et al., 2014). C3997Y (TGC to TAC), H4014N (CAC to AAC), C4017S (TGC to TCC), and C4032R (TGC to CGC) variants are the PrimeSTAR Mutagenesis Basal Kit. (TaKaRa) For easier fluorescence imaging analysis, wild-type and mutant mst-3×FLAG were cleaved at NheI and NotI sites, and pIRESpuro4 n-mCherry (Dr. Shigeo Murata, Graduate School of Pharmaceutical Science, provided by The University of Tokyo) The multi-cloning site (MCS) of this plasmid was used to match the open reading frame of vector-encoded mCherry and sub-cloned mst-3xFLAG. It was previously modified (the modified MCS sequence following mCherry was: SEQ ID NO: 1) and cleaved with XbaI and NotI prior to subcloning.peGFP-N1-ATGL was obtained from Dr. Hidde Ploegh ( Whitehead Institute, Massachusetts Institute of Technology) TIP-47/PLIN3-GFP was provided by Dr. Stefan Honing (Institute of Biochemistry I, University of Cologne) siRNA targeting human mysterin is Purchased from Invitrogen (#1: PNPLA2HSS125980 (3_RNAI); #2: PNPLA2HSS183700 (3_RNAI); #3: PNPLA2HSS183701 (3_RNAI)).
[antibody]
The mouse anti-mysterin monoclonal antibodies (1D6 and 1C9) were prepared as described previously (Kotani et al., 2017) and provided by Dr. Seiji Takashima, Graduate School of Medicine, Osaka University). Anti-PLIN3 antibody (Santa Cruz), anti-ATGL antibody (Santa Cruz for immunostaining; Cell Signaling for immunoblotting), anti-AUP1 antibody (Atlas Antibodies), anti-tubulin antibody (Millipore), anti-GAPDH antibody (Hy Test) , And anti-FLAG M2 antibody (Sigma) were used.
[CRISPR/Cas9 system]
Short double-stranded DNAs (SEQ ID NOS: 2 and 3) targeting the coding region of human mysterin were determined using CRISPR Search and Design Tool (Thermo Fisher), and pSpCas9(BB)-2A-Puro (PX459). V2.0 (provided by Dr. Feng Zhang; Addgene plasmid #62988). The vector was transfected into HeLa cells and maintained in the presence of 1 μg/mL puromycin for 7 days. The surviving cells were diluted and cultured, and the cells forming colonies were isolated and confirmed (#1 and #2 in FIG. S7A).
[Cell sorting]
Cell sorting was performed using the Lipid Droplet Isolation Kit (Cell Biolabs) according to the manufacturer's instructions. In FIG. 5H, 1/200 (total volume), 1/60 (lipid droplet; LD), 1/200 (cytoplasm), and 1/200 (membrane/cytoskeleton), and in FIG. Volume), 1/30 (LD), 1/250 (cytoplasm), and 1/250 (membrane/cytoskeleton) in FIG. 5J, 1/200 (total volume), 1/30 (LD), 1 /200 (cytoplasm) and 1/200 (membrane/cytoskeleton) were subjected to immunoblot analysis.
[Immunoblot]
Samples were prepared as previously described using cell fractionation procedures or directly from cells lysed with lysis buffer containing 1% NP-40. Samples were separated by SDS-PAGE using 5% or 3% to 10% gradient acrylamide gel (ATTO) and applied to PVDF membrane (Merck Millipore, Hessen, Germany) at 75V, 4°C for 1.5 hours. It was transcribed. The membrane was blocked with Blocking One (Nacalai Tesque) at room temperature for 1 hour, and the primary antibody (almost used at 1/200) and the secondary antibody diluted with Can Get Signal (TOYOBO) (1/200) were used. (Approx. used at 1000). The detection was performed by a chemiluminescence method.
[Immunostaining]
The cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 minutes at room temperature, permeabilized with 50 μg/mL digitonin for 5 minutes, and then washed with PBS. After incubation in Blocking One for 1 hour at room temperature, cells were sequentially incubated with primary antibody and fluorescently labeled secondary antibody, with a PBS wash after each incubation. Lipid droplets (LD) were stained with BODIPY 493/503 added at the same time as the secondary antibody and LipiDye (Funakoshi) added at the same time as the secondary antibody or after cell sorting. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). Fluorescent images were acquired on a confocal microscope (LSM 700, Carl Zeiss) at room temperature using ZEN software.
[FRAP measurement in living cells]
FRAP measurements were performed on a confocal microscope (LSM 700, Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4NA oil immersion objective (Carl Zeiss) using ZEN software. GFP-ATGL and PLIN3-GFP in HeLa cells were excited at 488 nm and photobleached at 488/555 nm. A traveling beam splitter (MBS405/488/555/639) was used. GFP or mCherry fluorescence was collected at 493-1000 nm or 578-1000 nm, respectively, using a variable second dichroic beam splitter. The size of the pinhole was set to 69 μm for GFP and 79 μm for mCherry. The scan size for the X and Y axes was 512 pixels. The image acquisition time interval was set to 10.0 seconds. The photobleaching time was 15 seconds. The relative fluorescence intensity (RFI) at time t was measured using ZEN software, and the following formula [1]:
Was calculated using. In equation [1], I _BL (t) and I _bg (t) are the intensities measured in the photobleaching region and the background region, respectively, at time t. Also in the formula [1], I′ _BL and I′ _bg are the fluorescence intensities before the photobleaching in the fading region and in the background region, respectively. _I'BL and _I'bg were measured in the cell free area.
The FRI recovery curve is the Origin 2017 software (OriginLab Corp) one-component exponential recovery model, ie, the following equation [2]:
Fit. In equation [2], I(t) is the intensity at time t and I ₀ is the baseline. In equation [2], A is an exponential recovery coefficient and k is a recovery rate. The maximum recovery rate (I _max ) is defined as I ₀ +A. The half-recovery time is calculated as -(ln 0.5)/k. In the analysis of PLIN3 and ATGL (FIGS. 7-1I and 7-1J and FIGS. 7-2K and L), the minimum decrease in fluorescence intensity immediately after photobleaching (baseline) was PLIN3-GFP fixed with 4% paraformaldehyde. Determined using expressing cells. The FRI was normalized to the lowest fluorescence intensity (Normalized RFI) to directly show the moving parts.
[Quantification of triglyceride (TG) by colorimetric analysis]
3×10 ⁶ HeLa cells were suspended in 250 μL PBS containing 1% Triton X-100. The supernatant was subjected to TG quantitative analysis (Serum Triglyceride Quantification Kit, CELL BIOLABS, INC.) by colorimetric analysis. The TG densities shown in Figure 6 are in the supernatant.
[Quantitative and statistical analysis]
The number and relative area of LD (and its associated protein) were analyzed using ImageJ (Wayne Rasband and NIH). All data were expressed as mean±SEM. Statistical significance was assessed using Student's t test. p<0.05 was considered statistically significant.

Results and Discussion In order to elucidate the intracellular distribution of mysterin, high resolution fluorescence microscopy using mysterin having mCherry at the N-terminus (mCherry-mst) was performed. Transiently expressed mCherry-mst showed a diffuse distribution in the cytoplasm in HeLa cells, and partially exhibited a donut-shaped pattern with a diameter of about 1 μm (see FIG. 1B). This aspect was reminiscent of medium-sized intracellular vesicle structures such as lysosomes, endosomes, and LDs. Of these, LD is a major fat storage site present in many tissues and a source of metabolic energy and fatty substances (Walther and Farese, 2012). This small droplet organelle is a phospholipid monolayer that often leaves a link between the water-insoluble core consisting mainly of neutral lipid triglycerides (TGs) and the ER that encloses it, and adipogenic, lipolytic, or regulatory functions. It is composed of a set of functional surface proteins (see FIG. 1C). Fluorescent staining of neutral lipids with BODIPY 493/503 revealed that mCherry-mst covered the LD surface in HeLa cells (see FIG. 1D). This fluorescent staining was more stable when oleic acid (OA; a major fatty acid in vivo) was added to HeLa cells, human embryonic kidney 293T (HEK293T) cells, and human hepatocellular carcinoma HepG2 cells. It was detectable (conditions with predominant lipogenesis; see Figures 5A-C). In addition, these cell lines and human adipocytes express mysterin endogenously (see FIG. 5D), although LD localized mysterin is also detectable (see FIGS. 1E and 1F; previously reported). (Kobayashi et al., 2015; Ohkubo et al., 2015) and as reproduced in FIGS. 5E and F, further good visualization of endogenous mysterin resulted in increased expression upon interferon stimulation. I need). Intracellular sorting supported that overexpressed and endogenous mysterins were distributed in the cytoplasm and LD in HEK293T and HepG2 cells (see Figures 5G-J). Therefore, mysterin is a protein that localizes to LD.

Approximately two localization pathways have been proposed for proteins that localize in LD: via the junction site of ER-LD (type I) and directly from the cytoplasm (type II and others) (Kory et al. al., 2016). Some proteins through the former pathway are first incorporated into the ER membrane by a hydrophobic hairpin motif and then transported to LD in a COPI mechanism-dependent manner (Soni et al., 2009; Wilfling et al. , 2014). The LD distribution of mCherry-mst was not sensitive to Brefeldin A (BFA), an inhibitor of the COPI mechanism (FIG. 1G), in contrast to ATGL, which shows a BFA-sensitive LD distribution. Yes (see Figure 5K). These results suggest that mysterin is not an LD protein with a type I hairpin motif. Rather, the considerable distribution of mysterin in the cytoplasm (see, eg, FIGS. 1B and 5H) and the complete absence in the membrane fraction (see FIGS. 5H-J) indicate that mysterin is targeted directly from the cytoplasm to LD. Suggest. Furthermore, mCherry-mst showed a considerable amount of fluorescence recovery (FRAP) after photobleaching in LD (see Figure 1H). This suggests that molecular exchange occurs between LD-localized mCherry-mst (photobleached) and cytoplasmic mCherry-mst (photoactive). The relatively large maximal recovery rate and short half-recovery time of mCherry-mst in LD compared to other LD proteins (ie ATGL, PLIN3/TIP-47, and AUP1) resulted in localization of mysterin to LD. Is reversible and rapid (FIGS. 4L and M). These results indicate that mysterin likely shuttles directly and rapidly between the cytoplasm and LD.

Cells transiently overexpressing mCherry-mst formed LD more extensively than untransfected cells (see Figure 2A). Quantitative analysis of LD revealed that the number of LD and the area occupied by LD were significantly increased in mysterin overexpressing cells (see FIG. 2B). In contrast, loss of mysterin protein by CRISPR/Cas9-mediated knockout (KO) or siRNA-mediated knockdown caused a significant reduction in LD even with the addition of OA (FIGS. 2C, 2D, and FIG. See 6C; validation of knockout and knockdown is shown in Figures 6A and B). A decrease in TG content in the KO cell line was also confirmed by quantification of TG by colorimetric analysis (see Figure 6D). These observations suggest that mysterin is involved in maintaining cellular fat stores.

The amount of neutral lipids in cells is largely determined by the balance of lipogenesis and lipolysis in cells, either or both of which may be regulated by mysterin. In other words, mysterin may promote lipogenesis and/or suppress lipolysis, increasing fat storage. Re-examine the effect of mysterin on LD in the presence of an inhibitor of lipogenesis (triaxin C; an inhibitor of fatty acid acyl CoA synthase) or an inhibitor of lipolysis (orlistat; a general lipase inhibitor), and It was determined if the process was affected by mysterin. Even when the rate-limiting step of TG synthesis was blocked by adding triaxin C, the effect of overexpression of mCherry-mst on LD enhancement was maintained (see Figures 2E and 2F). This suggests that the effect of mysterin on LD is largely independent of lipid synthesis. In contrast, inhibition of TG recruitment by orlistat largely negated the negative effect of mysterin knockout on LD (see Figures 2G and 2H). This suggests that mysterin increases LD mainly by interfering with the process of lipolysis.

Lipolysis of TG is stepwise promoted by three LD surface lipases, ATGL, HSL (hormone-sensitive lipase), and MGL (monoacylglycerol lipase) (Walther and Farese, 2012). Among these, ATGL is the rate-limiting and most influential lipase ((Zimmermann et al., 2004; Zechner et al., 2017). Cells overexpressing mCherry-mst were identified as LD. Although there was a marked reduction in associated ATGL (FIG. 3A, cells shown at o/e), many untransfected cells showed LD highly coated by ATGL (FIG. 3A, Consistent with this, mysterin KO cells showed a significant increase in ATGL attached to LD (see FIGS. 3B and 3C), whereas in KO cells, ATGL itself. There was no change in protein levels (see Figure 7-1A), therefore mysterin interfered with the distribution of ATGL, but not the total amount of ATGL in cells, and other LD proteins such as PLIN3 and AUP1 were identified as mCherry-mst. Partial colocalization (see Figures 3D and 7-1B-D), but ATGL did not show such colocalization with mysterin (see Figure 3E). Greater differences were observed below: mysterin always co-localized (100%) with overexpressed PLIN3 and AUP1 (see FIGS. 7E and F) but never (0%) with overexpressed ATGL. Therefore, it can be said that the mysterin-removing effect on LD surface proteins is specific to at least some extent, and the LD distribution of ATGL is mainly due to inflow and outflow from LD. Therefore, the disappearance (efflux) of ATGL in LD was monitored by selectively inhibiting the influx of ATGL into LD by the addition of BFA, and then, in mysterin KD cells, ATGL was lost. No clear delay was found in the efflux of E. coli (see Figure 7-1G), suggesting that mysterin removal of ATGL was not due to accelerated ATGL efflux from LD. We also confirmed the recovery (influx) of ATGL after removal.HeLa cells were pretreated with BFA for 24 hours to completely remove ATGL from LD.Recovery of ATGL after removal of BFA was more pronounced in mysterin KD cells. It was remarkable (see FIG. 7-1H), which suggests that mysterin removes ATGL by preventing the influx of ATGL into LD. It was evaluated more accurately by P measurement. The recovery of fluorescence after photobleaching of GFP-ATGL was markedly increased in mysterin KO cells, indicating that the molecular exchange of ATGL in LD is faster in mysterin KO cells (FIGS. 7-11 and 7-). 1J). This means that either influx or efflux of ATGL in LD and both influx and efflux are earlier in KO cells. Given that removal of mysterin positively affects ATGL accumulation in LD, influx rather than ATGL efflux is thought to be predominantly accelerated in KO cells (ie, endogenous mysterin induces ATGL in LD). Probably lowering the ATGL in LD by blocking the influx of). In contrast, PLIN3-GFP showed similar fluorescence recovery curves in wild cells and in mysterin KO HeLa cells, suggesting that the mysterin exclusion effect is not non-specific. Yes (see FIGS. 7-2K and L). Surprisingly, the specific exclusion event that occurs between mysterin and ATGL was not unidirectional. Overexpression of ATGL reversely removed mysterin from LD (see Figure 7-2M), but no such effect was observed with overexpressed PLIN3 or AUP1 (Figures 7-1E and 7-1F). reference). Therefore, mysterin and ATGL are likely to compete specifically with each other for LD localization (see Figure 7-2N). However, no physical interaction between mysterin and ATGL was observed (see Figure 7-20). This suggests that mysterin and ATGL may interfere with each other via a third factor (eg, competition for a common anchor protein that promotes localization of both proteins to LD). Mysterin knockout significantly destabilized LD, as shown in FIG. However, this effect was abolished by the additional knockdown of ATGL by siRNA (FIG. 3F; knockdown efficiency is shown in FIG. 7-2P). These results suggest that the stabilizing effect of LD by mysterin is due to the specific removal of the rate-limiting lipase, ATGL, from LD.

Mysterin exerts ATPase activity and ubiquitin ligase activity on the two AAA+ domains and the RING finger domain, respectively (Liu et al., 2011; Morito et al., 2014). Therefore, the involvement of these activities in LD formation was verified. FIG. 4A illustrates the enzymatic mutations used in this study. A1A2 has two essential mutations in the ATP binding motif (A motif) and B1B2 has two essential mutations in the Mg binding motif (B motif) that is essential for ATP hydrolysis. Therefore, these mutants either lose ATP binding activity (A1A2) or lose ATP hydrolytic activity (B1B2) (Morito et al., 2014). None of these mutants with mCherry at the N-terminus were localized in LD and dispersed throughout the cytoplasm (Fig. 4B). This indicates that the localization of mysterin to LD is essentially related to the ATP binding activity and the hydrolysis activity. Considering the analogy with certain AAA+ proteins (eg, catanine and Vps4), mysterin association and dissociation with LD is probably synchronized with the cycle of ATP binding and hydrolysis.

A mutant lacking the RING finger ubiquitin ligase domain (ΔRING) also altered the distribution. However, that aspect was different from that observed with the ATPase mutant. The ΔRING mutant with mCherry at the N-terminus (mCherry-ΔRING) showed an abnormal aggregate-like pattern and lost LD localization capacity in many cells (-80%) (left panel of FIG. 4C). The ΔRING mutant, which was successfully localized in LD, retained an exclusive effect on ATGL (see Figure 8A). This suggests that removal of ATGL does not occur via ubiquitination of ATGL by mysterin. Rather, mysterin ubiquitin ligase activity appeared to be involved in its maintenance or regulation for proper distribution into LD. Previous findings that the mysterin ubiquitin ligase activity is exerted on itself (self-ubiquitination) and is lost in the ΔRING mutant (Liu et al., 2011) have shown that a model of mysterin self-maintenance or self-regulation Can support.

As expected, the distribution of mysterin in LD and its fat stabilizing effect were interrelated. The A1A2 and B1B2 mutants did not show a positive effect on LD (Fig. 4D). The ΔRING mutant shows an aggregate-like pattern (agg) but loses its positive effect on LD, while the mutant that retained the LD localization (LD) found in a small number of cells remained constant. The LD stabilizing effect was shown (FIG. 4D). Therefore, it can be said that these two enzyme activities of mysterin are involved in the fat stabilizing function.

The involvement of the RING finger domain in the metabolic function of mysterin is particularly important considering the etiology of Moyamoya disease (MMD). Four mutations of conserved cysteine/histidine residues in the RING finger domain were recently identified in MMD cases of Caucasians (C3997Y, H4014N, C4017S, and C4032R) (see Figure 4E) (Guey et al. , 2017; Raso et al., 2016; Cecchi et al., 2014). It was revealed that all four mutants with a 3xFLAG tag at the C-terminus do not lose LD localization and show aggregate-like structure formation that is very similar to that observed with the ΔRING mutant. (See Figure 4F). These mutants also lost the metabolic effect (Fig. 4G). This suggests a pathogenic association of abnormal LD metabolism in Caucasian MMD. On the other hand, R4810K of C-terminal region without putative functional structure, which is a mutant in other MMD, and non-conserved residue in RING finger domain, East Asian/Caucasian Species D4013N ((Liu et al., 2011) caused no detectable abnormalities (see Figures 8B and C). Deletion of such effects was due to these putative structurally non-essential Possibly due to location.In addition, the heterogeneous intensity of the molecular effects of these variants may explain these diverse pathogenic effects in MMD.

Discussion A detailed analysis of the intracellular distribution of mysterin reveals that mysterin is targeted to LD and that it functions as a positive regulator of cellular fat storage by selective removal of ATGL from LD. It was Multistep TG recruitment initiated by ATGL occupies a key position in cellular/life energy metabolism and is regulated by multiple factors/pathways, including those stimulated by catecholamines and insulin. (Zechner et al., 2017). The knockdown/knockout of endogenous mysterin caused a significant loss of fat in the cells, suggesting that mysterin is involved in maintaining fat stores in vivo.

The LD localization and fat stabilizing effects of mysterin require its enzymatic activity. Unlike many AAA+ proteins that form stable oligomers (presumably hexamers), mysterins dynamically change their oligomeric state (presumably hexamers). Mysterin formed an oligomer upon binding to ATP in the first AAA+ module and dissociated upon hydrolysis of another ATP in the second AAA+ module (see Figure 8D) (Morito et al., 2014). The mysterin A1A2 and B1B2 mutants have lost their ATP-binding and hydrolytic activities, respectively, and partially terminate the oligomerization/dissociation cycle in the monomer and oligomer states, respectively (Morito et al., 2014). ). Localization to LD was commonly impaired in these mutants (see Figure 4B), which indicates that mysterin localization to LD was static (ie, monomeric or oligomeric). ), suggesting that it is regulated through a dynamic cycle of ATP binding/hydrolysis. The mysterin oligomerization cycle was similar to some exceptional AAA+ members. Katanin and Vps4, while bound to ATP, oligomerize on microtubules and target protein complexes such as ESCRT-III, respectively (see Figure 4H) (Ogura and Wilkinson, 2001). It then mediates the degradation of these target complexes during ATP hydrolysis and then returns to the diffusing monomeric form (see Figure 4H). Mysterin can similarly assemble and dissociate on the target LD (or on surface proteins) in conjugation with the binding/hydrolysis cycle of ATP (see Figure 41). In tannin and Vsp4, they directly bind to these substrate complexes and mediate their dissociation, but no physical interaction between mysterin and ATGL was observed (see FIG. 7-2O). ), there was no apparent enhancement of the already existing ATGL efflux in LD (see Figure 7G). Even if mysterin exerts a similar activity to katanin and Vsp4, its direct target should not be ATGL. A rational scenario is that mysterin and ATGL compete with each other for binding to a common anchor protein that promotes localization of both proteins to LD (see Figure 7-2N). Mysterin can affect the putative anchor protein using its AAA+ activity, preventing the influx of ATGL into LD. Conversely, ATGL can monopolize the anchor protein, removing mysterin from the LD.
Targeting mysterin to LD is affected by its ubiquitin ligase activity. The ΔRING mutant and Caucasian Moyamoya disease cysteine/histidine mutants showed substantial elimination from LD, indicating the formation of aggregate-like patterns (FIGS. 4C and 4F). This suggests that mysterin's own ubiquitin ligase activity (self-ubiquitination) exerts a self-maintaining or self-regulatory function similar to that found in other ubiquitin ligases (Baldridge and Rapoport, 2016).

From the perspective of Moyamoya disease, it was difficult to predict this finding in advance. Previous etiology studies did not assume a relationship between obesity and dyslipidemia and other metabolic disorders (Scott and Smith, 2009). Histopathological studies showed thickening of vascular smooth muscle cells and thrombus formation in the vascular lumen in lesions, but atherosclerotic changes were not typically observed (Scott and Smith, 2009). ). Therefore, Moyamoya disease has not been previously thought to be closely associated with lipid metabolism. However, the findings that multiple moyamoya disease mutations in the RING finger domain impaired the metabolic function of mysterin suggest a potential link between moyamoya disease and adiposity. A range of human disorders, including cardiovascular disorders, are associated with dysfunction and/or dysregulation of LD in various cells/tissues (often caused by mutant LD proteins) (Krahmer et al., 2013). However, it is not known which cells/tissues (eg, vascular endothelium, smooth muscle, endothelial progenitor cells, or immune cells) form a major disorder in Moyamoya disease (Scott and Smith, 2009). Aberrant aggregate-like structures of mysterin mutants are thought to exert cytotoxicity as seen in many neurodegenerative diseases (Labbadia and Morimoto, 2015), and the association between moyamoya disease and dyslipidemia has been mostly reported. Based on its absence, it is thought that this cell disorder is the main cause of moyamoya disease.
Unlike the prominent localization/dysfunction of Caucasian cysteine/histidine variants in the RING finger domain, an East Asian R4810K variant and an East Asian/Caucasian D4013N variant at a putative nonessential site The mutant did not clearly change the metabolic function of mysterin. This fact may explain the degree of genetic penetrance of these variants. Carriers of R4810K and D4013N are susceptible to moyamoya disease but do not always develop (ie, have very low/imperfect penetrance), but carriers of cysteine/histidine variants are always moyamoya. Disease (ie, its genetic penetrance is very high/complete) (Raso et al., 2016; Liu et al., 2011; Kamada et al., 2011; Guey et al., 2017; Cecchi et al., 2014). R4810K and D4013N may exert only moderate or very slight effects on the metabolic function of mysterin and, as has been repeatedly discussed in previous etiology and genetic studies, R4810K and D4013N exert additional environmental and Only if there is a genetic factor (for example, inflammation), it is thought that the cause of the onset of moyamoya disease (Scott and Smith, 2009; Koizumi et al., 2016).

Our results show that mysterin wraps lipid droplets, ubiquitous organelles specialized for triglyceride storage, and stabilizes the lipid droplets through removal of ATGL lipase from the lipid droplet surface. Was done. Both ATPase and ubiquitin ligase activities are required for the correct localization of mysterin to lipid droplets. Localization of mysterin to correct lipid droplets is markedly impaired by Caucasian moyamoya disease mutations in the ubiquitin ligase domain. From these findings, it became clear that mysterin is a new component of the intracellular fat storage mechanism. In addition, it is suggested that the relationship between the onset of moyamoya disease and the formation of abnormal aggregate-like structure of mysterin caused by genetic mutation.

Claims (9)

A pharmaceutical composition comprising an inhibitor of mysterin, for use in treating Moyamoya disease or for promoting fat metabolism. The pharmaceutical composition according to claim 1, wherein the mysterin inhibitor is a mysterin activity inhibitor and/or expression inhibitor. The pharmaceutical composition according to claim 2, wherein the inhibitor of mysterin is an inhibitor of activity of mysterin. The pharmaceutical composition according to claim 3, wherein the inhibitor of mysterin is an AAA+ ATPase inhibitor. The pharmaceutical composition according to claim 3, wherein the inhibitor of mysterin is a ubiquitin ligase inhibitor. The pharmaceutical composition according to claim 2, wherein the mysterin inhibitor is a mysterin expression inhibitor. The pharmaceutical composition according to claim 6, wherein the expression inhibitor is selected from the group consisting of siRNA, shRNA or its expression vector, antisense oligonucleotide, and miRNA. Use as a model of abnormal fat metabolism in non-human mammals in which protein expression and/or function of mysterin is reduced, or as a model of individual disorder due to abnormal aggregate formation of mysterin. A method for screening a compound that regulates fat metabolism, or a compound that eliminates abnormal aggregate formation of mysterin, comprising:
A method comprising contacting a test compound with mysterin or a variant thereof.