CN112870358B - Use of deubiquitinase USP20 for reducing cholesterol synthesis and improving metabolic syndrome - Google Patents

Abstract

The invention provides application of an antagonist or an inhibitor of deubiquitinase USP20 protein or a coding gene thereof, wherein the antagonist or the inhibitor is used for preparing a medicine for reducing cholesterol synthesis and improving metabolic syndrome.

Description

Use of deubiquitinase USP20 for reducing cholesterol synthesis and improving metabolic syndrome

Technical Field

The invention relates to the technical field of biology, in particular to application of deubiquitinase.

Background

Cholesterol is the most abundant sterol in mammals. It plays a variety of roles including regulating membrane function¹Is a precursor of bile acid and steroid hormone synthesis²Covalent modification of Hedgehog and Smoothened proteins^3-5. Cholesterol and other lipid metabolism abnormalities are major risk factors for cardiovascular disease, non-alcoholic fatty liver disease (NAFLD), obesity, and diabetes⁶

It is estimated that an adult newly synthesizes or absorbs a total of about 1g of cholesterol per day from the intestinal tract⁷. The liver is the major organ for cholesterol biosynthesis and accounts for approximately 50% of the total individual production. Starting from acetyl-CoA, cholesterol is synthesized by approximately 30 reaction steps, where HMG-CoA reductase (HMGCR) is the rate-limiting enzyme catalyzing the conversion of HMG-CoA mevalonate. The biosynthesis of cholesterol is an energy and nutrient consuming processThe process. It requires 18 acetyl CoA, 18 ATP and 20 NADPH (NADH) molecules to produce a single molecule of cholesterol⁹. In order to avoid the toxicity of cholesterol excess to cell membranes, the biosynthesis of cholesterol is tightly regulated, mainly by two pathways: SCAP-SREBP pathway and HMGCR degradation pathway^10,11。

High levels of dietary cholesterol can inhibit cholesterol biosynthesis by the above mechanisms. However, in the history of evolution, the supply of food has never remained stable and the animals experienced cycles of famine (fasting) and satiation (feeding)¹². It has long been the key to the survival of mammals in hunger that inhibits cholesterol biosynthesis and increases its synthesis after eating. However, the mechanism of increased cholesterol biosynthesis caused by feeding is not clear.

Ubiquitin carboxyl-terminal hydroscope 20(UPS20) is a member of the deubiquitinase family. Human USP20 is 914 amino acids in length and 102,003Da in molecular weight. The protein sequence is shown in SEQ ID No. 1.

The gene ID of the humanized USP20 gene at NCBI is 10868, and the sequence of the coding region is shown as SEQ ID NO. 2.

HMGCR is mainly ubiquitinated by E3 ligase gp78 in liver, and is degraded by targeted proteasome^13-15。

Disclosure of Invention

The invention aims to provide application of an antagonist or an inhibitor of deubiquitinase USP20 protein or a coding gene thereof.

According to one aspect of the invention, the use of an antagonist or inhibitor of the deubiquitinase USP20 protein or of a gene encoding it for the preparation of a medicament and/or therapeutic agent for reducing feeding induced cholesterol synthesis and ameliorating metabolic syndrome.

In the use of the present invention, the antagonist or inhibitor may be selected from the following:

an agent that inhibits the enzymatic activity of USP 20;

an agent that reduces the expression level and phosphorylation level of USP20 protein;

substances that reduce or block USP20 binding to gp 78; and/or

A substance which reduces or blocks the expression of a gene encoding deubiquitinase USP 20.

In preferred embodiments, the antagonist or inhibitor includes, but is not limited to: antibodies or fusion proteins against the protein deubiquitinase USP20, antisense nucleic acids against the gene encoding deubiquitinase USP20, blockers or inhibitors of the USP20-gp78 signaling pathway.

More preferably, wherein the antagonist or inhibitor recognizes the Ser132 and/or Ser134 site of the USP20 protein.

Various inhibitors of the protein USP20 may be used in the present invention, including currently known and subsequently developed inhibitors, and a preferred inhibitor of the deubiquitinase USP20 protein is GSK2643943A ((E) -2-amino-6- (3-fluorostyryl) -1H-indole-3-carbonitrile).

In the use of the present invention, the drug and/or the therapeutic agent can be used for treating and/or preventing the following diseases: diet-induced weight gain, obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, non-alcoholic fatty liver disease, obesity and diabetes. Can be used for reducing serum and liver lipid levels, increasing insulin sensitivity, increasing energy expenditure, lowering cholesterol, and reducing triglycerides.

According to another aspect of the present invention, there is provided a use of deubiquitinase USP20 protein or a gene encoding the same, wherein the deubiquitinase USP20 protein or the gene encoding the same is used for screening drugs and/or therapeutic agents for diseases associated with eating-induced cholesterol synthesis and metabolic pathways. It is understood that substances which can inhibit or reduce or block the expression of the protein USP20 or the gene encoding the protein, or inhibit or block the signaling pathway of USP20-gp78 can be screened to determine the substances which can be used as candidate drugs or therapeutic agents for treating and/or preventing related diseases.

The present invention also provides a method for treating and/or preventing a disease, comprising administering an antagonist or inhibitor of deubiquitinase USP20 protein or a gene encoding the same or a pharmaceutical composition comprising the same to a subject, said disease and use being selected from the group consisting of: reducing serum and liver lipid levels, increasing insulin sensitivity, increasing energy expenditure, reducing cholesterol, reducing triglycerides, diet-induced weight gain, obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, non-alcoholic fatty liver disease, obesity and diabetes.

Statins are cholesterol-lowering drugs that are currently widely used clinically, and they are inhibitors of HMGCR enzyme activity. Although statins are effective in lowering blood cholesterol and treating cardiovascular and cerebrovascular diseases, statins cause new-onset diabetes and block adipocyte browning. Unlike statins, the present inventors have found that inhibitors of USP20 inhibit cholesterol synthesis primarily after eating, without altering the basal enzymatic activity of HMGCR. USP20 inhibitor not only reduces liver lipids and blood lipids, but also promotes increased energy metabolism. The results of our research show that the inhibition of USP20 can treat metabolic diseases such as obesity, cardiovascular and cerebrovascular diseases, hyperlipidemia, non-alcoholic fatty liver, diabetes and the like.

Drawings

FIG. 1 USP20 is essential for food-induced HMGCR elevation

(a) Immunoblotting examined the expression levels of HMGCR, FDFT1, LSS, and DHCR24 proteins in mouse liver. Starvation group mice were starved for 12h (n-5) before sampling, and feeding group mice were starved for 12 hours before feeding with high sugar low fat feed for 12 hours before sampling (n-5).

(b) Protein expression levels were quantified in panel (a). HMGCR, FDFT1, LSS and DHCR24 were all normalized to GAPDH. The mean value of each protein in starvation state was defined as 1.

(c) Real-time quantitative PCR was performed to determine the relative levels of liver mRNA levels in mice in the starved and refeed groups.

(d) Schematic diagram of in vitro deubiquitination experiment. Cholesterol starved CHO-7 cells were treated with 1. mu.g/ml 25-HC and 10. mu.M MG-132 for 2h to induce HMGCR ubiquitination, and the prepared membrane fraction was co-immunoprecipitated with HMGCR antibody-conjugated microbeads. The pellet samples were then incubated with the liver cytoplasm of starved or refeeded mice for 30 minutes at 37 ℃. After washing the samples, analysis was performed by western blotting with ubiquitin antibody or HMGCR antibody.

(e) In vitro deubiquitinating western blot results for HMGCR.

(f) The deubiquitinase USP20 can stabilize the HMGCR protein. Huh7 cells were transfected with the indicated plasmids, sterol-starved for 16 hours, and treated for 5h with or without 1. mu.g/mL 25-HC and 10mM mevalonate medium. Cells were collected and subjected to SDS-PAGE and immunoblot analysis.

(g) As shown in FIG. 1a, for 8 weeks old WT and littermate L-Usp20^-/-Mice (3 per group) were starved and refeeded. Liver samples were subjected to immunoblot analysis with the indicated antibodies.

All values are expressed as mean ± SEM. Data were analyzed by t-test. n.s. indicates no statistically significant difference. P <0.05, p <0.01, p < 0.001.

FIG. 2 glucose and insulin activate the mTOR pathway to phosphorylate the USP 20S 132 and S134 sites to regulate its function

(a) M199 Medium (containing 5.5mM glucose) for WT and L-Usp20^-/-Primary hepatocytes of mice. On day 1, cells were untreated or treated with additional 20mM glucose plus 10nM insulin. After 3 or 5 hours, cells were collected and immunoblotted.

(b) M199 Medium cultured WT mice in primary hepatocytes. On day 1, cells were transfected with the indicated plasmids and incubated in medium A (containing 5% LPDS, 1. mu.M lovastatin and 10. mu.M mevalonate) for 14 hours. Cells were pretreated for 2 hours in medium in the absence or presence of the indicated protein kinase inhibitor (Wort. wortmannin, 0.2. mu.M; Akti, Akt1/2 kinase inhibitor 10. mu.M; rapamycin 0.1. mu.M), and cells were treated with or without 1. mu.g/ml 25-HC plus 10mM mevalonate for 5 hours in the absence or presence of insulin and the indicated inhibitor. Immunoblots showed protein levels of HMGCR, USP20, p-AKT, p-mTOR, mTOR and actin.

(c) Huh7 cells were transfected with the indicated plasmids and after 16 h sterol starvation, treated with or without 1. mu.M of the AMPK inhibitor Dorsomorphin 2HCl and 5.5mM or 25.5mM glucose for 5 h. Immunoblots showed protein levels of HMGCR, USP20, p-AMPK, AMPK, p-mTOR, and actin.

(d) Huh7 cells were transfected with the indicated plasmids and after 16 hours of sterol starvation, cells were treated with or without 1. mu.M AMPK activator A-769662 in medium containing 25.5mM glucose, with or without 1. mu.g/ml 25-HC and 10mM mevalonate for 5 hours. Immunoblots showed protein levels of HMGCR, USP20, p-AMPK, AMPK, p-mTOR, and actin.

(e) Domain schematic of USP 20. The zinc finger ubiquitin binding domain (ZnF-UBP), the catalytic domain and DUSP are indicated. The two phosphorylation sites (S132 and S134) are red.

(f) In vitro kinase reactions showing phosphorylation levels of FLAG-labeled USP20 WT and mutant C154S after incubation with active mTOR (1362-terminal, active). Coomassie blue stained gels indicate the concentration of protein in each reaction. (G) livers were isolated from mice starved for 12 hours or fed high sugar low fat diet for 12 hours, respectively, and liver extracts were immunoprecipitated with USP20 endogenous antibody plus protein A/G beads. As shown, lysates were immunoblotted with pan-phosphorylated Ser/Thr antibody, phosphorylation-specific antibodies (p-S132 and S134), HMGCR antibody, and USP20 antibody.

(h) Huh7 cells were transfected with the indicated plasmids and 48 hours later, the cells were treated with or without 0.1. mu.M rapamycin for 2 hours. Cells were collected and whole cell lysates were Immunoprecipitated (IP) with anti-FLAG beads. Samples were subjected to immunoblot analysis with the indicated antibodies.

FIG. 3 phosphorylation of S132 and S134 of USP20 is crucial for food-induced increases in HMGCR

(a) Huh7 cells were transfected with the indicated plasmids and after 16 h sterol starvation, treated with or without 1. mu.g/ml 25-HC plus 10mM mevalonate for 5 h. Cells were collected for immunoblot analysis.

(b) WT, C154S and S132/S134A were tested for in vitro deubiquitinase activity by the Ub-AFC assay. NC: no protein was added. Western blot showed similar expression of purified protein.

(c) WT and gp 78-knock-out cells were transfected with the indicated plasmids, USP20 using FLAG beads IP. K89 and K248 are ubiquitination sites of HMGCR.

(d) Huh7 cells were transfected with the indicated plasmids and 48 hours later, the cells were lysed and incubated for 30 minutes in a reaction system with or without lambda phosphatase (lambda PPase). Samples were IP-labeled with FLAG beads and then immunoblotted.

(e) As depicted in FIG. 1a, for 8-week old male WT and littermate Usp20^KI/KI(S132A/S134A mutant form expressing USP20) mice (5 per group) were starved and refeeded. Liver samples were subjected to immunoblot analysis with the indicated antibodies.

(f) A simplified schematic diagram depicting the regulatory mechanism of the glucose and insulin signaling pathways for USP 20. Glucose and insulin signaling pathways activate mTOR, which phosphorylates S132 and S134 of USP20, USP20 in turn binds gp78 and increases stability of HMGCR.

FIG. 4L-Usp 20^-/-Mouse performance metabolism improvement

8-week old male WT and littermate L-Usp20^-/-Mice were randomized into groups (n ═ 6 per group), ad libitum on drinking water and high-carbohydrate high-fat (HFHS) diet (60% fat, 20% carbohydrate and 20% protein (w/w) for 23 weeks all data are presented as mean ± SEM<0.05，**p<0.01，***p<0.001。

(a) Body weight of each mouse after 23 weeks of feeding.

(b) Food intake of two groups of mice.

(c-d) body fat composition of 31-week-old mice was measured by NMR.

(e) Ratio of liver weight to body weight in 31-week-old mice.

(f) Liver sections (top) and White Adipose Tissue (WAT) (bottom) were stained with H & E, and liver sections (middle) were stained with oil red O to show lipid content, with a scale bar representing 50 μ M.

(g) Quantifying the size of the adipocytes in (f). The area of 15 arbitrary adipocytes (n-4) per mouse was analyzed by ImageJ software.

(h-j) Total Cholesterol (TC) and Triglyceride (TG) levels in serum and liver.

(k-l) FPLC analysis of cholesterol (j) and TG (k) in mouse sera.

(m) Glucose Tolerance Test (GTT) (left panel) and area under the curve (AUC) (right panel). The experiment was performed 14 weeks after HFHS diet.

(n) insulin resistance test (ITT) (left panel) and AUC (right panel). The experiment was performed after 15 weeks on HFHS diet.

(o, p) mice were tested for systemic oxygen consumption during the night and day cycles. The experiment was performed 16 weeks after HFHS diet.

(q) taking infrared thermography after eating again to measure the body surface temperature, and displaying as a representative image. The experiment was performed 22 weeks after HFHS diet.

(r) rectal temperature of mice after refeeding. The experiment was performed 22 weeks after HFHS diet.

(s) rectal temperature of mice at various times after cold stimulation (4 ℃). The experiment was performed 23 weeks after free HFHS feeding of the mice.

(t) detection of metabolites associated with the TCA cycle in the liver. The WT and the littermate L-Usp20^-/-(n-4) after starving mice for 12 hours and eating for 6 hours, the liver metabolite ratios were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS). By mixing L-Usp20^-/-Fold change was calculated by dividing mouse values by WT mouse mean. Data are mean ± SEM. P<0.05 (u, v) levels of succinic acid in mouse serum and liver after refeeding.

FIG. 5 USP20 inhibitor improves metabolism in mice

(a) Experimental protocol for inhibitor treatment. 8-week-old C57BL/6J mice were gavaged daily with 0 or 30mg/kg GSK2643943A (USP20 inhibitor) dissolved in 10% 2-hydroxypropyl- β -cyclodextrin for 13 days. GTT and metabolic cage assays were performed on day 7 and day 10, respectively.

(b) After 13 days of oral administration of the inhibitor, mice were starved and refeeded as described in figure 1 a. Western blot analysis of HMGCR, p-S132/134, USP20, FASN and actin in liver was performed using the indicated antibodies.

(c-d) TC and TG levels in mouse serum after treatment with GSK2643943A (G). TC and TG levels were measured after starvation and refeeding. V denotes the control group and G denotes the GSK2643943A treated group.

(e-f) mice were tested for systemic oxygen consumption during the night and day cycles.

(g-h) content of succinic acid in mouse serum and liver after treatment with vehicle or GSK 2643943A.

(i) Experimental protocol for oral administration of GSK2643943A in Diet Induced Obese (DIO) mice. 8-week old C57BL/6J mice were divided into two groups after 15 weeks feeding HFHS and orally administered with 0 or 30mg/kg GSK2643943A 2 for 2 weeks, respectively. GTT and metabolic cage assays were performed on day 7 and day 10, respectively.

(j) DIO mice orally administered with 0 or 30mg/kg GSK2643943A were tested for body weight.

(k-n) TC and TG content in liver and serum of DIO mice.

(o) liver sections (top) and White Adipose Tissue (WAT) (bottom) were stained with H & E. Scale bar represents 50. mu.M.

(p) quantifying the size of the adipocytes in (o). The area of 15 arbitrary adipocytes (n-4) per mouse was analyzed by ImageJ.

(q) Glucose Tolerance Test (GTT) (left panel) and area under the curve (AUC) (right panel).

All data are expressed as mean ± SEM. Statistical significance analysis of variance was performed using either the t-test or Tukey's HSD test. n.s. indicates no statistically significant difference. P <0.05, p <0.01, p < 0.001.

Description of the expanded figures

Figure 6 in vitro ubiquitination assay of HMGCR.

(a) Schematic diagram of in vitro ubiquitination experiment. Membrane fractions were prepared from sterol-starved CHO-7 cells to provide non-ubiquitinated HMGCR and E3 complexes. Liver cytoplasm was prepared from mouse liver under starvation or refeeding conditions. The membrane fraction was incubated with E1, UBE2G2, ATP, FLAG-ubiquitin, cytoplasm and hydroxycholesterol (25-HC) for 30 min at 37 ℃. Endogenous HMGCR was IP with HMGCR antibody in IP buffer. Western blotting was performed with FLAG antibody or HMGCR antibody.

(b) Results of in vitro ubiquitination experiments with HMGCR as performed in (a).

FIG. 7 screening of DUB expression library and identification of USP20 as a regulator of HMGCR

(a) Schematic diagram of screening process. Huh7 cells were seeded at 4X 10 on day 0⁵Cells/60 mm dish, in medium A containing 10% (vol/vol) FBS. On day 1, cells were co-transfected in 3ml of medium A containing 1. mu.g pCMV-HMGCR-T7, 30ng pCMV-Insig-1-Myc and 0.3. mu.g Dubs. The total DNA amount of each sample was adjusted to 2. mu.g/disc by adding pcDNA3 empty vector. 8 hours after transfection, cells were incubated in 3ml of medium A supplemented with 10% (vol/vol) FBS. On day 2, cells were washed with Phosphate Buffered Saline (PBS) and then switched to medium A containing 5% LPDS, 1. mu.M lovastatin and 10. mu.M mevalonate, after incubation for 16 hours at 37 ℃, cells were cultured for 5 hours with or without 1. mu.g/ml 25-HC plus 10mM mevalonate, and 2 dishes of cells were pooled, harvested, lysed and immunoblotted.

(b-i) immunoblot analysis was performed with 1. mu.g/ml monoclonal anti-T7 IgG (for HMGCR) and 1. mu.g/ml monoclonal anti-Myc IgG (for insight-1) as described above.

FIG. 8 screening of DUB expression libraries

(a-g) the experimental screen described in FIG. 7a was performed to screen for DUB that modulates USP 20.

FIG. 9 description of Deubiquitinase USP20

(a) USP20 reduced sterol-induced HMGCR ubiquitination. CHO-7 cells were transfected with the indicated plasmids, starved for 16 h of cell sterols as shown in FIG. 7a, and then treated with the indicated concentrations of 25-HC in the presence of 10. mu.M MG132 for 2 h. Cells were collected, lysates immunoprecipitated with anti-T7 beads, and the pellet was immunoblotted with HA antibody and HMGCR antibody.

(b) CHO-7 cells were transfected with the indicated siRNAs. After 48 hours, as shown in FIG. 7a, the cells were sterol-starved for 16 hours, then treated with the indicated concentration of 25-HC plus 10mM mevalonate for 5 hours, the cells were harvested and immunoblotted.

(c) Quantification of the level of HMGCR protein in (b).

(d) WT USP20 and catalytically inactivated mutant USP20(C154S) protein were purified from HEK293T cells. The deubiquitinating activity of the USP variants was determined by Ub-AFC assay. Negative Control (NC) indicates no protein added. Western blot showed similar expression of purified protein.

(e) WT USP20 and USP20(C154S) proteins expressed and purified by HEK293T cells were immunoblotted after incubation with the substrate (Ub-2) di-ubiquitin (K6), (K11), (K27), (K29), (K33), (K48) or (K63), respectively, for 2 hours at 37 ℃.

FIG. 10 characterization of liver-specific Usp20 knockout mice

(a) Tissue expression profile of mouse USP20 protein level.

(b) Tissue expression profile of mouse Usp20 mRNA levels.

(c) Schematic strategy for targeting the Usp20 gene knockout.

(d) WT and L-Usp20^-/-Western blot analysis of different tissues of mice.

(e-g) free-feeding WT and L-Usp20^-/-(n-5) body weight, food intake and serum AST level of mice. All values are expressed as mean ± SEM. Data were analyzed by t-test. n.s. indicates no statistically significant difference.

FIG. 11 starvation and refeeding treated WT and L-Usp20^-/-Mouse assay

(a) Starved and refeeded WT and litter L-Usp20^-/-Analysis of metabolic parameters in mice. Each value represents the mean ± SEM of 5 mice. Under the same treatment conditions, WT and L-Usp20^-/-The level of statistical significance between mice was analyzed using the t-test. + p<0.05，++p<0.01，+++p<0.001. "a" indicates a comparison of WT mice eating and hungry; "b" denotes L-Usp20^-/-Comparison of mice fed and starved. "c" denotes starved L-Usp20^-/-Comparison to starved WT mice; d denotes L-Usp20 fed^-/-Comparison with fed WT mice.

(b) Graph (a) relative amount of mouse liver mRNA was detected by qPCR. The target genes are involved in cholesterol synthesis, SREBP pathway, fatty acid synthesis, LXR pathway and fatty acid oxidation pathway.

FIG. 12 hepatic HMGCR protein levels are regulated by glucose and insulin

(a) WT mice starved overnight were injected intraperitoneally with 2mg/g glucose or 0.75mU/g insulin or 2mg/g glucose plus 2mg/g insulin. After 3 hours, mouse livers were extracted separately and the lysates were analyzed by 8% SDS/PAGE. HMGCR and USP20 protein levels were analyzed by immunoblotting, actin as a control.

(b) Effect of insulin and glucose on mouse liver Usp20 and Hmgcr mRNA levels.

(c) After the mouse primary hepatocytes were not treated (-) or (+) with 25.5mM glucose or 10nM insulin for 3 hours or 5 hours, the cells were collected and subjected to immunoblot analysis with the indicated antibodies.

(d) Effect of insulin and glucose on levels of Usp20 and Hmgcr mRNA in mouse primary hepatocytes. Statistical analysis was performed on b and d using single variance analysis and multiple comparison tests by Bonferroni. Data represent mean ± SEM. n.s. indicates no statistical difference p <0.05, p <0.01

(e) Huh7 cells were transfected with the indicated plasmids, 6 hours later, the cells were incubated in media containing 5% LPDS, 1. mu.M lovastatin and 10. mu.M mevalonate, 14 hours later, the cells were pretreated for 2 hours in media without (-) or with (+) the indicated protein kinase inhibitor (Wort. wortmannin, 0.2. mu.M; Akti, Akt1/2 kinase inhibitor 10. mu.M; rapamycin 0.1. mu.M), the cells were treated with 1. mu.g/ml 25-HC plus 10mM mevalonate for 5 hours, and then immunoblotted with the indicated antibodies.

FIG. 13 identification of phosphorylation site of USP20 and analysis of gp78-USP20 interaction

(a) Schematic representation of the identification of phosphorylation site of USP 20. Huh7 cells were transfected with pCMV-USP20-3 xFlag. After 6 hours of culture at 37 ℃, the cells were incubated in a low glucose medium containing 10% FBS for 16 hours, the cells were switched to low glucose or low glucose supplemented with 20mM glucose (high glucose) for 12 hours, and then the cells were collected. The USP20 protein was immunopurified using anti-Flag M2 sepharose beads and competitively eluted with Flag peptide fragments. The eluted protein was subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS).

(b) The first 10 phosphorylated USP20 peptide fragments. H: high glucose; l: low glucose.

(c) Mass spectrometry analysis indicated that USP20 was modified by phosphorylation at sites S132 and S134.

(d) Schematic representation of gp78 and its various truncated forms. Binding of gp78 variants to USP20 was summarized as positive (+) or negative (-). (e) Huh7 cells were transfected with the indicated plasmids, after sterol starvation and treatment with 1. mu.g/ml of 25-HC and 10mM mevalonate for 3 hours, cell lysates were IP-treated with anti-Flag M2 agarose beads. Western blotting was performed with the indicated antibodies.

(f) CHO cells were transfected with plasmids expressing the gp78 variant and USP20, and whole cell lysates were incubated with anti-Flag M2 agarose beads for IP USP 20. Western blotting was performed with the indicated antibodies.

(g) Huh7 cells were transfected with the indicated plasmids for 48 hours and cell lysates were IP-treated with anti-Flag M2 agarose beads. Western blotting was performed with the indicated antibodies.

FIG. 14 Usp20^KI/KICharacteristics of the mouse

(a-f) on 12 week old male WT and littermate Usp20^KI/KIMice were starved and refeeded.

(a-e) serum is tested for total cholesterol, total triglycerides, free fatty acids, insulin and glucose levels. Data were analyzed by t-test. P<0.05，**p<0.01，***p<0.001. Each value represents the mean ± SEM of the data of 5 mice. (f) Starvation and refeeding treated WT and Usp20 by qPCR detection^KI/KIRelative amount of mouse liver mRNA. Target genes include cholesterol synthesis, SREBP pathway, fatty acid synthesis, LXR pathway and fatty acid oxidation pathway.

FIG. 15L-Usp 20^-/-And Usp20^KI/KIAnalysis of mice

(a, b) phenotype of 31-week-old mice. 8-week old male WT mice and littermate L-Usp20^-/-Mice were randomized (n ═ 6 per group) on free water and HFHS diets for 23 weeks.

(c-i) 8 week old Male WT and Homoppet Usp20^KI/KIMice were randomized (n ═ 5 per group) and were on free water and HFHS diet for 14 weeks. Each timeValues represent mean ± SEM. Two-way analysis of variance (Tukey post hoc test) was used. P <0.05, p <0.01, p < 0.001. n.s. indicates no statistically significant difference.

(c) Phenotype of 22 week old mice.

(d) Body weight of each mouse after 14 weeks of HFHS feeding.

(e, f) the total body fat composition of 22-week-old mice was measured by NMR. All values are expressed as mean ± SEM. Data were analyzed by t-test and n.s. represent statistically no significant difference.

(g) Liver weight of 22 week old mice.

(h) And (5) GTT analysis. Area under GTT analysis curve (AUC) is shown on the right.

(i) Insulin resistance test (ITT) analysis. The area under the ITT analysis curve (AUC) is shown on the right.

Data are presented as mean ± SEM. Statistical significance of GTT and ITT was tested by Tukey's HSD. P <0.05, p <0.01, p < 0.001.

(j-t) 8 week old Male WT and Homopolar L-Usp20^-/-Mice were randomized (n ═ 6 per group) and were on free water and HFHS diet for 23 weeks. All values are expressed as mean ± SEM. Data were analyzed by t-test. n.s. indicates no statistically significant difference.

(j, k) WT and L-Usp20 measured in metabolic cages after 16 weeks of HFHS diet^-/-Respiratory Entropy (RER) and movement profile during the night and day cycle of the mice.

(L-t) after 23 weeks of HFHS diet, WT and L-Usp20^-/-Mice were tested for serum levels of epinephrine, norepinephrine, triiodothyronine (T3), thyroxine (T4), secretin, Thyroid Stimulating Hormone (TSH) and FGF 21.

FIG. 16 analysis of mice orally administered with USP20 inhibitor GSK2643943A

(a) Chemical structure of GSK 2643943A.

(b) GSK2643943 inhibits the in vitro deubiquitinating enzyme activity of USP 20. NC: no USP20 protein.

(c) GTT analysis of mice in figure 5 a. Area under GTT analysis curve (AUC) is shown on the right. Values are mean ± SEM. Data were analyzed by two-way variance using Tukey's HSD test. P <0.05, p <0.01, p < 0.001.

(d-e) ALT and AST levels in mouse serum in FIG. 5 a. Values are mean ± SEM, and the t-test assesses significant differences. n.s. indicates no statistically significant difference.

(f) Figure 5i food intake of mice during treatment. Statistical significance was assessed using the t-test. n.s. indicates no statistically significant difference.

(g, h) fat content, lean content of the mice of FIG. 5 i. Values are mean ± SEM. Data were analyzed by two-way variance using Tukey's HSD test.

(e-1) the liver weight, brown fat weight, heart weight and kidney weight of the mice in FIG. 5i were examined on day 14. All values are expressed as mean ± SEM and data are analyzed by t-test. n.s. indicates no statistically significant difference. Western blot analysis of HMGCR, USP20, phosphorylated AKT and total AKT in liver lysates after 14 days treatment of DIO mice with vehicle or GSK 2643943A.

(n, o) metabolic cage assay the average systemic oxygen consumption of mice in FIG. 5 i.

(p-s) the serum ALT, AST, creatinine and TNF alpha content of the mice are detected.

(t) Gene expression levels of mouse liver and WAT of FIG. 5i were examined by qPCR.

All values are expressed as mean ± SEM. The o-t data were analyzed by t-test and n.s. indicates no statistically significant difference. P <0.05

Detailed Description

Based on the teachings herein, it will be understood by those skilled in the art that the expression "antagonist or inhibitor of the deubiquitinase USP20 protein or its encoding gene" in the present invention refers to a molecule or substance that antagonizes or inhibits (e.g., reduces, inhibits, decreases, degrades or delays) the activity of the deubiquitinase USP20 protein or its encoding gene, and also includes molecules or substances that antagonize or inhibit the binding of USP20-gp 78. In particular embodiments, the "reduction or decrease" refers to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% decrease in USP20 protein activity or the amount of expression of the USP20 encoding gene as compared to the original USP20 protein activity or the amount of expression of the USP20 encoding gene, i.e., no expression of the USP20 encoding gene at all or a complete loss of USP20 protein activity. The 'medicament' comprises an antagonist or an inhibitor of deubiquitinase USP20 protein or a coding gene or RNA thereof and a pharmaceutical composition containing the antagonist or the inhibitor and pharmaceutically acceptable auxiliary materials, and the 'therapeutic agent' can be a therapy, such as gene therapy, or other substances which are beneficial to improving disease conditions, such as food, health products, additives and the like.

The invention will be described in detail but not limited by the following description of preferred embodiments of the invention:

materials and methods

Mouse

Usp20-floxed mice were constructed by the Nanjing biomedical research institute. The sgRNA is targeted between introns 6 and 7 and between introns 8 and 9 by the CRISPR/Cas9 system, mediates cleavage of Cas9 endonuclease, and results in insertion of LoxP sites between introns 6 and 7 and between introns 8 and 9, respectively, by homologous recombination. The mouse is hybridized with albumin-Cre tool mouse to obtain liver specificity USO20 knockout mouse (L-Usp 20)^-/-)。

Mice carrying the USP 20S 132A & S134A mutations were constructed by CRISPR/Cas 9-mediated genome editing techniques (Cyagen Biosciences). Briefly, grnas (ggc CGT TAC CTC GAGs GCT TCA gg) were inserted into gRNA cloning vectors. Donor oligonucleotides were designed and synthesized simultaneously, including the S132A (TCT to GCT) mutation site and the S134A (TCT to GCT) mutation site. Then, mRNA and gRNA generated by in vitro transcription of Cas9, and donor oligonucleotide were injected into mouse zygotes, and mice with USP 20S 132A & S134A point mutations were screened.

In the experiment, the genotype was Usp20^flox/floxThe littermates of (a) were used as wild type control mice. All mice are raised in a ventilation cage to ensure the environmentNo pathogen, 12 hours light/dark 12 hours, and feeding standard normal feed. In starvation and refeeding experiments, mice were divided into two groups: starvation and refeeding groups. The starved group mice were starved for 12 hours, and then the fed group was starved for 12 hours and then fed with high-sugar low-fat feed (Research Diets, D12450B) for 12 hours. In diet-induced mice obesity experiments, 8-week-old male mice were fed high-sugar, high-fat diet (Research Diets, D12468B), and experimental analysis was performed after 23 weeks. The changes in oxygen consumption, respiratory entropy and movement of mice were measured using mouse metabolism cages (CLAMS, Columbus Instruments, Columbus, OH) and monitored over 2 days. A small animal body composition analyzer was used to detect changes in mouse body fat. The anal temperature of the mice was detected by a rectal probe connected to a digital thermometer (physiotemp, NJ). The infrared images were taken with an infrared camera SC1000E (FLIR Systems inc., Wilsonville, OR). All animal experiments strictly comply with the ethical and protection related regulations of the welfare of the experimental animals of the university of the country and Wuhan.

Materials and plasmids

MG-132, ubiquitin activating enzyme (E1), recombinant Di-Ubiqnitin chain of human origin (K6, K11, K27, K29, K33, K48 and K63), ubiquitin AFC (Ub-AFC) and FLAG-ubiquitin were purchased from Boston Biochem; dorsomorphin 2HCl (AMPK inhibitor) and A-769662(AMPK activator) were purchased from Selleck Chemicals; lovastatin is purchased from Shanghai medicinal grain in China; mevalonate and 25-hydroxycholesterol were purchased from Sigma; biological grade [ 2 ]³H]Water (100mCi/ml, ART 0194) was purchased from American radio laboratory chemicals. Lipoprotein deficient serum (LPDS, d) was prepared from newborn calf serum by ultracentrifugation in this laboratory>1.215g/ml)。

The following plasmids were constructed by standard molecular cloning techniques: pCMV-HMGCR-T7, encoding full-length hamster HMGCR, with a T7 tag. pCMV-Insig-1-Myc, encoding human-derived Insig-1, carrying the Myc tag. pEF-FLAG-Ubiquitin, coding human Ubiquitin, with FLAG label. pCMV-gp78-Myc, encoding human gp78, with a 5 × Myc tag. pCMV-USP20-FLAG, encoding humanized USP20, with a 3XFLAG tag.

GSK2643943A is available under the chemical name (E) -2-amino-6- (3-fluorostyryl) -1H-indole-3-carbonitrile from MedChemexpress under the catalog number HY-111458.

EXAMPLE 1 cell culture

CHO-7, Huh-7 and HEK293T cells at 37 ℃ and 5% CO₂The environment of (2) is grown as a monolayer. Huh-7 and HEK293T cells were maintained in Medium A (DMEM with 100units/mL penicillin and 100mg/mL streptomycin sulfate) and supplemented with 10% Fetal Bovine Serum (FBS). CHO-7 and CHO-gp78-KO cells were maintained in 5% FBS supplemented medium B (a 1: 1 mixture of DMEM and Ham's F-12 medium containing 100units/mL penicillin and 100mg/mL streptomycin sulfate). Cholesterol starvation medium C was obtained by supplementing medium A or medium B with 5% lipoprotein deficient serum (LPDS), 1. mu.M lovastatin and 10. mu.M mevalonate. Primary mouse hepatocyte cultures were supplemented with medium D (M199) 5% FBS, 100units/mL penicillin and 100. mu.g/mL streptomycin sulfate.

Example 2 immunoblotting

The collected cells or tissues were lysed in RIPA lysis buffer supplemented with protease inhibitors. The RIPA lysis buffer contained 50mM Tris-HCl, pH 8.0, 150mM NaCl, 2mM MgCl2, 1.5% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate. The protease inhibitor comprises 10. mu.M MG-132, 10. mu.g/ml leupeptin, 1mM phenylthiolactonyl fluoride, 5. mu.g/ml pepstatin, 25. mu.g/ml N-acetylleucol-norleucinol and 1mM dithiolitol. Protein concentration of lysates was determined using BCA method (Thermo Fisher Scientific). Protein samples were incubated with membrane lysis buffer (62.5mM Tris-HCl, pH 6.8, 15% SDS, 8M urea, 10% glycerol and 100mM DTT) and 4 Xloading buffer (150mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 6% 2-mercaptoethanol and 0.02% bromophenol blue) for 30 min at 37 ℃. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes. Blocking with TBS containing 0.075 % Tween 20 and 5% skim milk for 1 hour and incubating overnight with the indicated primary antibody at 4 ℃. Followed by 3 washes in TBST, incubation with HRP-conjugated secondary antibody (1: 5000) at room temperature for 1 hour, followed by 3 washes with TBST. Finally, detection was performed using Pierce ECL Plus Western blot substrate (Thermo Fisher Scientific).

The primary antibody information used in the experiment was as follows: anti-phospho (Ser/Thr) phe (#9631), anti-phospholated-AKT (8200), anti-AKT antibody (4691), anti-phospholated-mTOR (5536), and phor-AMPK α (2535) were purchased from Cell Signaling Technology. anti-Flag (F1804) and anti-Beta Actin (# A1978) were purchased from Sigma. anti-AMPK alpha (10929-2-AP), anti-FDFT1(13128-1-AP), anti-LSS (18693-1-AP), anti-DHCR24 (10471-1-AP), anti-FASN (66591-1-Ig) and anti-GAPDH (10494-1-AP) were purchased from Proteintetech. Specific anti-phosphorylated Ser132 and Ser134 antibodies were obtained by immunizing rabbits with phosphopeptide fragments. The horseradish peroxidase-conjugated goat anti-mouse (115-.

Example 3 immunoprecipitation

Cells were washed twice with ice-cold PBS buffer before being collected in 600. mu.l of cold lysis buffer (PBS and containing 0.5% digitonin, 5mM EGTA, 5mM EDTA, protease and phosphatase inhibitors). After centrifugation (12,000g, 10 min) of the whole cell lysate, the precipitate was discarded, 60. mu.l of the Supernatant was taken as the whole cell lysate control (Input fraction), 500. mu.l of the Supernatant was mixed with 30. mu.l of primary antibody, incubated at 4 ℃ for 2 hours with rotation, centrifuged at 1000g for 5 min, 60. mu.l of the Supernatant was taken as the Supernatant control (Supernatant fraction), the remaining Supernatant was discarded, washed 3 times with lysis buffer, 10 min each time and mixed with rotation. The pellet was finally collected and 120. mu.1 SDS PAGE Loading buffer was added, labeled pellet fraction. Then, each component is detected by Western blot.

Example 4 preparation of mouse liver cytoplasm fraction

Male mice (30-35 g) fed under normal conditions were starved and refeeded. One group of mice was starved for 12 hours and the other group was starved for 12 hours before eating the high sugar, low fat feed for 12 hours. Mouse livers were perfused with 0.9% (w/v) NaCl via the hepatic portal vein at room temperature. The livers were then excised, cut into small pieces, and homogenized with ice-cold buffer (50mM HEPES-KOH, pH 7.2, 250mM sucrose, 70mM potassium acetate, 5mM EGTA, 2.5mM magnesium acetate). The buffer contained protease inhibitors (20. mu.M leupeptin, 5. mu.g/ml pepstatin A, 25. mu.g/ml N-acetylleucine-leucine-norleucine, 1mM dithiothreitol). All subsequent steps were carried out at 4 ℃. The supernatant was centrifuged at 20,000g for 20 minutes, 186,000g for 1 hour and 186,000g for 45 minutes in that order. Mu.l of the supernatant was taken for BCA protein quantification. The final supernatant was cytoplasmic fraction (15-20mg protein/ml). The final supernatant was aliquoted and stored at-80 ℃.

EXAMPLE 5 preparation of cell membrane fraction

CHO-7 cells were inoculated into medium B containing 10% FBS (8X 10 per 10cm dish)³Individual cells). At 5% CO₂After 12 hours of culture at 37 ℃, adherent cells were washed once with PBS and cultured overnight in medium C at 37 ℃. After 16 hours, two 10cm dishes were collected and resuspended in 500. mu.l (10mM HEPES/KOH, pH 7.6, 1.5mM MgCl2, 10mM KCl, 5mM EDTA, 250mM sucrose, protease inhibitor) buffer. The cell suspension was lysed 15 times through a 22.5 gauge needle and centrifuged at 1,000g for 10 min at 4 ℃. The supernatant was then removed at 4 ℃ at 2X 10⁴g centrifuged for 15 minutes to prepare the membrane fraction. The fractions were stored at-80 ℃.

Example 6 in vitro assay for Deubiquitination of HMG-CoA reductase

First, a reaction buffer was prepared comprising 25mM Hepes-KOH, 115mM potassium acetate, 5mM sodium acetate, 2.5mM MgCl2, 0.5mM EGTA, 2mM Hepes-KOH at pH 7.3, 1mM magnesium acetate, 1mM ATP, 30mM creatine phosphate and 0.05mg/ml creatine kinase at pH 7.3. Then 0.1mg/ml ubiquitin, 1.6. mu.g/ml E1, 1.6. mu.g/ml E2, 10. mu.g/ml 25HC and 10. mu.g/ml cholesterol were added. A reaction buffer solution with a certain volume is taken, and a cell membrane component and a mouse liver cytoplasm component are added to prepare a reaction system with a medium volume of 0.3 ml. Incubate at 37 ℃ for 30 minutes. Followed by centrifugation at 13,200rpm for 10 minutes at 4 ℃. Lysates were pre-reacted with 3. mu.g rabbit IgG and 40. mu.l protein A/G plus-Sepharose (sc-2003, Santa Cruz Biotechnology) for 1 hour at 4 ℃ followed by immunoprecipitation with 6. mu.g rabbit polyclonal HMGCR antibody and 100. mu.l Ig A/G Sepharose beads overnight at 4 ℃. After subsequent 5 washes with immunoprecipitation buffer containing protease inhibitors, it was boiled with 2 Xloading buffer for 10 minutes at 95 deg.C, centrifuged at 12,000rpm for 1 minute, and the supernatant was mixed with an equal volume of HMGCR lysis buffer and incubated at 37 deg.C for 30 minutes. Immunoprecipitated aliquots were subjected to SDS-PAGE, transferred to PVDF membrane, and subjected to immunoblot analysis.

Example 7 in vitro ubiquitination and deubiquitination assays

Wild-type and enzyme-active mutant proteins of USP20 were overexpressed and purified in HEK293T cells. All sorts of Ub2 were incubated with equal samples of USP20-WT and USP20-C154S in 30. mu.l of deubiquitinating buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA, 5mM dithiothreitol) for 2 hours at 37 ℃. Further incubated with 10. mu.l of 4 Xloading buffer at 37 ℃ for 30 minutes and immunoblotted with the antibody against P4D 1. DUB activity was determined by measuring the increase in fluorescence upon cleavage of ub-AFC. Purified USP20-WT and USP20-C154S (. about.50 nM) were first added to 200. mu.l of deubiquitinating buffer (50mM Tris-HCl pH 7.4, 20mM potassium chloride, 5mM magnesium chloride, 1mM DTT, including 0.5. mu. MUb-AFC), respectively, and incubated at 37 ℃ for 1 hour. The fluorescence intensity was measured using a spectrometer with excitation and emission wavelengths set at 400 and 505nm, respectively.

Example 8 in vitro phosphorylation assay

FLAG-tagged USP20-WT and USP20-S132A/S134A fusion proteins were first purified from HEK 293T. In vitro phosphorylation assay reaction system 20. mu.l was added 150ng of fusion protein, 20ng of active mTOR (Millipore, 14-770) and ATP in reaction buffer (25mM HEPES pH 7.4, 50mM KCl, 5mM MgCl2 and 5mM MnCl2, 50mM) and incubated at 30 ℃ for 30 min. The reaction was then stopped by adding 5. mu.l of 4 Xloading buffer, boiled at 95 ℃ for 10 minutes and analyzed by SDS-PAGE.

Example 9 glucose tolerance and insulin tolerance test

Prior to the study, mice were starved for 16 hours (GTT) or 4 hours (ITT). In the GTT experiment, mice were injected intraperitoneally with glucose (2g/kg body weight). For the ITT experiments, mice were injected intraperitoneally with insulin (0.75U/kg body weight). Tail vein blood glucose levels were measured at 0, 15, 30, 60 and 120 minutes post injection using the Onetouch Ultra blood glucose monitoring system (Johnson), respectively.

Example 10 blood and liver chemistry analysis

Blood is collected by eyeballs to obtain serum for detecting total cholesterol and triglyceride; the liver was homogenized, and the supernatant was collected for lipid extraction to obtain total liver cholesterol, and total liver TG (Kehua, China) levels were determined according to the instructions. Free fatty acid levels were measured with a kit (NEFA, Wako, Japan). The level of insulin in serum was measured by elisa (ezassay). A kit (NJJCBIO, China) built by Nanjing is used for detecting the ALT and AST levels of serum. Serum levels of T3, T4, TNF- α, TSH, secretin, epinephrine, norepinephrine, and creatinine were measured using an ELISA kit available from Cloud Clone Corporation.

Example 11 TCA metabolite analysis

Liver tissue (approximately 20mg) and 20 μ L of internal standard solution were extracted with 400 μ L of pre-cooled 80% aqueous methanol (containing 2.5% formic acid) at 45Hz for 90s (3 times) using a tissue lysis instrument, followed by 10 sonication cycles (1 min sonication, 1min pause). After centrifugation for 10 minutes (12,000rpm, 4 ℃), the supernatant was collected and the above extraction process was repeated twice. The resulting three supernatants were combined, the methanol removed in vacuo and lyophilized. The dried extract of each sample was re-dissolved in 80% aqueous methanol (100. mu.l) for LC-MS/MS analysis.

UPLC-MS/MS analysis was performed on an Agilent UPLC-MS/MS system consisting of a 1290UPLC system and an Agilent 6470 triple quadrupole mass spectrometer (Agilent Technologies, USA). Mu.l of the extract was loaded for analysis. Chromatography was carried out on a Waters ACQUITY UPLC HST 3 column (2.1X 100mm, 1.8 μm) at 40 ℃ in a gradient of 6 minutes (0-1min 1% B,1-3min from 1% B to 15% B,3-4min from 15% B to 95% B,4-6min 95% B) with a flow rate of 0.5mL/min, using solvent A (water containing 0.1% formic acid) and B (methanol containing 0.1% formic acid). Electrospray ionization was performed on the nebulizer using N2 in negative ion mode at a flow rate of 10L/min and a temperature of 315 deg.C at a pressure of 50 psi. The temperature of the sheath gas is 350 ℃, and the flow rate is 10L/min. The capillary was set at 4000V. Multiple Reaction Monitoring (MRM) has been used to quantitatively screen fragment ions.

Peak determination and peak area integration were performed using MassHunter workstation software (Agilent, version b.08.00) with manual inspection of the automatic integration and correction if necessary. The target peak areas were obtained by correcting and calculating the reaction ratios with the appropriate Internal Standard (IS) throughout the analysis.

Results and analysis

Example 12 HMGCR expression is regulated by starvation-refeeding

To explore the mechanism of cholesterol biosynthesis regulation under starvation and feeding conditions, we examined the expression of several cholesterol synthases in the liver, including HMGCR, farnesyl diphosphate farnesyltransferase 1(FDFT1), lanosterol synthase (LSS) and 24-dehydrocholesterol reductase (DHCR 24). After the hunger over night, the mice are fed with the high-sugar low-fat feed, the cholesterol content of the feed is extremely low, and the interference of negative feedback regulation is reduced. Compared to the starvation state, HMGCR protein expression increased by about 20-fold under fed conditions (fig. 1a, b). Whereas other enzymes such as FDFT1, LSS and DHCR24 only increased by about 1.5-2 fold (fig. 1a, b). The mRNA expression of all these genes increased by about 2-4 fold (FIG. 1 c). The magnitude of the increase in HMGCR protein was more pronounced than the magnitude of the increase in HMGCR mRNA (fig. 1 b). These results indicate that post-prandial liver increases cholesterol synthesis by inducing an increase in HMGCR protein, which plays an important role in the regulation process through post-translational modification.

HMGCR is mainly ubiquitinated by E3 ligase gp78 in liver, and is degraded by targeted proteasome^13-15. We tested whether the ubiquitination modification of proteins plays a role in HMGCR protein regulation by in vitro ubiquitination experiments. As shown in FIG. 6a, membrane fractions providing non-ubiquitinated HMGCR and enriched by Insigs and gp78 were first isolated from CHO-7 cells lacking sterols¹³The E3 complex of composition. Isolating the cytoplasmic fraction from the liver of starved or fed mice, incubating E1, UBE2G2, ATP, Flag-ubiquitin, 25-hydroxycholesterol (25-HC) with the two fractions, and performing an in vitro ubiquitination assay (FIG. 6a)¹⁶. 25-HC could significantly induce ubiquitination of HMGCR in the presence of liver cytoplasm (fig. 6 b). Notably, the mice in the starvation group were refed to HMGCR in the groupThe level of ubiquitination was significantly reduced (fig. 6 b). These results indicate two possibilities: 1) the starved mice had factors in the liver cytoplasm that enhanced the activity of E3. 2) The liver cytoplasm of the mice in the feeding group had stronger Deubiquitinase (DUB) activity.

Next, we performed in vitro deubiquitination experiments to distinguish these two possibilities (fig. 1 d). CHO-7 cells were cholesterol starved and treated with 25-HC (inducing ubiquitination of HMGCR) and MG132 (inhibiting protein ubiquitination degradation)¹⁷Immunoprecipitated (IP) HMGCR, and incubated with the liver cytoplasm of starved or fed mice (fig. 1 d). HMGCR ubiquitination levels did not change after incubation with the liver cytoplasm of starved mice, but were significantly reduced after incubation with the liver cytoplasm of fed mice (fig. 1 e). Taken together, these results indicate that feeding-induced animal hepatocytes contain stronger DUB activity, protecting HMGCR from ubiquitin-proteasome degradation. This increased food-induced stability of HMGCR may have important effects on the storage of energy and increase of cholesterol synthesis in animals that eat.

Example 13 Deubiquitinase USP20 stabilizes HMGCR protein

Approximately 90 DUBs in the human genome¹⁸. To determine which might be involved in HMGCR degradation, we performed a functional screen by co-expressing a single DUB with HMGCR and Insig-1, where Insig-1 is necessary for sterol-induced HMGCR protein degradation¹⁹. The results show that deubiquitinase 20(USP20) significantly inhibited degradation of HMGCR (fig. 1f, fig. 7a), whereas none of the other DUBs showed any significant effect (fig. 7, fig. 8). 3). Human USP20 is a cytoplasmic protein of 914 amino acids involved in antiviral responses, DNA damage checkpoints and other functions^20-22. Overexpression of USP20 reduced sterol-induced HMGCR ubiquitination (fig. 9 a). In contrast, knockdown of USP20 accelerated degradation of HMGCR (fig. 9b, c). Although other proteins have been reported to be substrates of USP20, such as beta-catenin and Claspin, they are not related to HMGCR or cholesterol biosynthesis^21，23，24

To determine whether USP20 interaction with HMGCR was dependent on its deubiquitinaseActivity, we constructed a mutant with inactivated enzyme catalytic function USP20(C154S)²⁵And the wild type and mutant proteins were purified. First, it was shown by ubiquitin ub-AFC experiment that the deubiquitinating enzyme activity of WT was normal, while the enzyme activity of C154S mutant was lost (FIG. 9 d). USP20 hydrolyzed only K48 and K63 linked substrates of ubiquitin bis (Ub2), while no significant activity was observed for K6-, K11-, K27-, K29-or K33 linked substrates (fig. 9 e). Meanwhile, the results indicate that the catalytically inactive mutant USP20(C154S) fails to stabilize HMGCR (fig. 1f), thus confirming that the deubiquitinase activity of USP20 is necessary for its function of stabilizing HMGCR.

Example 14 liver-specific knockout of Usp20 inhibited food-feed induced increases in HMGCR

USP20 was highly expressed in mouse liver (fig. 10a, b), suggesting an important function in liver. We constructed liver-specific Usp20 knockout mice (L-Usp 20)^-/-)。L-Usp20^-/-The mice and wild type littermates thereof were fed with normal feed, and had no significant difference in body weight and food intake level, and no liver damage was indicated by aspartate Aminotransferase (AST) in serum (FIG. 10 e-g). Next, mice were treated with starvation-refeeding, consistent with the results in fig. 1a, feeding significantly increased HMGCR protein levels in the liver of WT mice (fig. 1 g). In contrast, feeding hardly increased L-Usp20^-/-HMGCR levels in mice (fig. 1 g). WT and L-Usp20^-/-Levels of insulin signaling such as phosphorylated Akt (p-Akt) and Insig-2 and FASN in response to starvation-refeeding regulation were similar in mice (FIG. 1 g). These data indicate that USP20 is specifically involved in meal-induced HMGCR stabilization without affecting the insulin pathway.

Figure 11a summarizes various metabolic parameters of mice in starvation and refeeding states. And previous reports^14,26In agreement, WT mice had elevated serum cholesterol, triglyceride, glucose and insulin levels and reduced free Fatty Acid (FA) levels in the fed state. In contrast, in L-Usp20^-/-In mice, these lipid levels, as well as glucose, were significantly reduced, while the serum insulin levels were nearly identical between the two groups under the same treatment (fig. 11 a). In WT and L-Usp20^-/-The gallbladder being fixed between miceThe mRNA levels of the alcohol synthesis genes Srebp2 and Scap were similar (fig. 11 b). L-Usp20^-/-Has lower Srebp1c mRNA than WT mice, and the expression of SREBP1c target gene (such as FA synthesis gene) is reduced. LXR target genes include Abca1, Abcg1, Abcg5 and Abcg8 at L-Usp20^-/-Is also down-regulated (FIG. 11b), probably as a result of a reduced production of sterol intermediates of the cholesterol synthesis pathway, which are capable of activating LXR and Srebp1c and the expression of their target genes^27,28

Example 15 glucose and insulin synergistically regulate USP20 function

Which signal pathways are involved in the regulation of HMGCR by USP 20? It is well known that the most significant changes in glucose and insulin levels following ingestion are²⁹. To examine the effects of glucose and insulin, WT mice starved overnight were injected intraperitoneally with glucose or insulin. When only glucose or insulin was present, HMGCR protein levels increased (4.9-11.9 fold), the increase upon co-injection was more pronounced (-20 fold) (fig. 12a), while mRNA levels were only weakly increased (fig. 12 b). The results were similar to the fed conditions (FIGS. 1 a-c). Furthermore, in primary hepatocytes of WT mice, glucose and insulin significantly increased the protein level of HMGCR with little effect on its mRNA level (fig. 2a and fig. 12c, d). In contrast, primary hepatocytes lacking Usp20 were non-responsive to insulin and glucose (fig. 2 a).

Insulin exerts its regulatory function by binding to the Insulin Receptor (IR), followed by phosphorylation of the insulin receptor substrate protein (IRS)³⁰. Phosphorylated IRS activates phosphoinositide 3 kinase (PI 3K)/AKT/rapamycin (mTOR) pathways, which play an important role in insulin metabolism³¹. To illustrate whether USP 20-mediated stability of HMGCR was involved in the insulin pathway, we overexpressed USP20, HMGCR and Insig-1 in isolated mouse primary hepatocytes. The results show that USP20 partially prevented sterol-induced HMGCR degradation (fig. 2b, lanes 3, 4). Insulin enhanced the effect of USP20 in stabilizing HMGCR (figure 2b, lanes 5, 6). However, Wortmannin (PI3K inhibitor), AKTi (AKT inhibitor) and rapamycin (mTOR inhibitor)³²Inhibits USP 20-mediated accumulation of HMGCR (FIG. 2b, lanes 7-12). In human liver cancer Huh7 cell, it was also confirmedSimilar effects of these inhibitors (fig. 12 e). These results indicate that USP20 may be involved in the mTOR signaling pathway.

It is well known that AMPK inhibits endogenous mTORC1 activity in response to glucose levels³³. By examining the effect of glucose on USP20 mediated HMGCR stabilization, the results indicated that USP20 effectively inhibited sterol-induced HMGCR degradation under high glucose conditions (fig. 2c, lanes 3, 4), but not under low glucose conditions (fig. 2c, lanes 5, 6). USP20 restored stabilization to HMGCR under low glucose conditions when treated with the AMPK inhibitor Dorsomorphin (fig. 2c, lanes 7, 8). In contrast, AMPK activator a-769662 inhibited USP 20-mediated stabilization of HMGCR under high glucose conditions (fig. 2 d). Taken together, these results indicate that USP 20-mediated HMGCR stabilization is modulated by glucose and insulin signaling pathways.

Example 16 mTOR phosphorylates S132 and S134 of USP20

The previous data indicate that USP20 stabilized HMGCR in fed state, but neither starvation-refeeding nor glucose-insulin altered USP20 levels in the liver (fig. 1g and fig. 12 a). How glucose and insulin regulate USP 20. It is known that stability of HMGCR by kinase cascade reaction and insulin/glucose signaling shows different results under different glucose conditions (fig. 2c), so we compared phosphorylation levels of USP20 in low (5.5mM) or high (25.5mM) sugar treated Huh7 cells (fig. 13 a). Quantitative mass spectrometry detected multiple phosphorylated peptide fragments of USP20, in which Ser132 and Ser134 phosphorylation were only detectable under high glucose conditions (fig. 13b, c and fig. 2 e). Indicating that the two sites can be detected in mTORC1 phosphorylation proteomics research^34,35. Whether insulin signaling activates mTOR in turn phosphorylates USP20 and promotes stabilization of HMGCR.

To test this hypothesis, we generated site-specific phosphoantibodies against USP20 phosphorylated Ser132 and Ser134(pS132/pS134) and performed in vitro kinase experiments with activated forms of mTOR. The results indicate that WT USP20 is significantly more phosphorylated by mTOR than the USP20 mutant (S132A/S134A) (fig. 2 f). Feeding significantly increased phosphorylation of endogenous USP20 (figure 2 g). In addition, rapamycin reduced the phosphorylation level of WT USP20, whereas USP20(S132A/S134A) was almost free of phosphorylation modifications (fig. 2 h). Taken together, USP20 is a genuine substrate for mTOR and is phosphorylated predominantly at the S132/S134 site.

Next, we investigated how USP 20S 132/S134 phosphorylation promoted HMGCR stabilization. USP20(S132A/S134A), which was not modified by phosphorylation, failed to stabilize HMGCR, although it had DUB activity similar to WT USP20 (FIG. 3 a). We next examined whether phosphorylation affected the interaction of the USP20-HMGCR complex. Figure 3c shows that HMGCR binds to WT USP20 in a dose-dependent manner, whereas USP20(S132A/S134A) does not bind. USP20 and HMGCR were unable to interact in gp78 knockout cells (fig. 3c), indicating that gp78 is involved as an intermediate in the interaction of USP20 and HMGCR. Gp78 contains an N-terminal multi-transmembrane domain (1-308a.a.) and a C-terminal cytoplasmic domain, consisting of a catalytic RING domain, a CUE motif and an E2 binding domain (fig. 13 d). The gp78-USP20 interaction was not affected by sterols (FIG. 13e), and the experimental results showed that the region required for gp78 binding to USP20 was the 383-578 segment of gp78 (FIG. 13 f). IP experiments showed that WT USP20 interacts with gp78, while the S132A/S134A mutation significantly reduced the USP20-gp78 interaction (FIG. 13 g). In addition, lambda dephosphorylatase treatment reduced phosphorylation modification of USP20, and also significantly reduced binding of gp78 to USP20 (fig. 3 d). Taken together, experimental data indicate that mTOR is able to phosphorylate S132/S134 of USP20, and that these two sites are required for USP20 to bind gp 78.

Example 17 analysis of mice typed in (KI) USP20(S132A/S134A)

To investigate the in vivo function of S132/S134 of USP20 modified by mTOR phosphorylation, we constructed mice with mutations at the S132A and S134A sites of USP20 protein (Usp20)^KI/KI) And mice were starved-refeeded. Following feeding, hepatic HMGCR protein levels were significantly increased in WT mice, but at Usp20^KI/KIThere was only a slight increase in mice (fig. 3 e). Phosphorylation of S132/S134 of USP20 was also lost (FIG. 3 e). Usp20 compared to WT mice^KI/KIMice had decreased serum levels of TC and TG under starvation and refeeding conditions (graph)14a, b). Although WT and Usp20^KI/KIMice showed similar levels of free FA and insulin in starvation and refeeding states (fig. 14c, d) and fasting blood glucose levels (fig. 14e), but the glucose level in the fed state Usp20KI mice was significantly lower than WT mice (fig. 14 e). Usp20^KI/KILiver Gene expression levels in FIG. 14f, results are in comparison with L-Usp20^-/-Mice were similar (fig. 11 b). These results demonstrate that mTOR-mediated phosphorylation of USP20 is critical for food-activated HMGCR stabilization and cholesterol synthesis. From the results of fig. 2 and 3 and fig. 13, we propose a working model as shown in fig. 3 f. Feeding induces insulin and glucose signaling pathways in the liver to activate mTOR, thereby phosphorylating S132 and S134 of USP20, and phosphorylated USP20 binds gp78, stabilizing HMGCR by deubiquitination, thereby increasing cholesterol biosynthesis.

Example 18L-Usp 20^-/-And Usp20^KI/KIMetabolic index of mice

In recent years, increasing research results have demonstrated the importance of the mTOR signaling pathway in nutrient perception and storage. Our results indicate that USP20 may be a key mTOR downstream protein for efficient use of liver nutrition and energy. To further understand the function of USP20 in metabolism, we performed L-Usp20^-/-Mice were given a high-sugar, high-fat diet (HFHS). Although L-Usp20^-/-And WT mice had no significant difference in food intake and lean muscle mass (FIG. 4b, c), but L-Usp20^-/-The mice lost weight, but fat mass and liver weight were significantly reduced (fig. 4a, d, e. fig. 15 a). The liver of the L-Usp 20-/-mice was red (FIG. 15 b). Hematoxylin and eosin (H) by liver and White Adipose Tissue (WAT)&E) And oil Red O staining, L-Usp20^-/-The size of adipocytes in WAT was reduced in mice and fat accumulation in liver was reduced (fig. 4f, g). Furthermore, L-Usp20^-/-TG levels in mouse liver (fig. 4h) and TC and TG levels in serum (fig. 4i, j) were decreased, cholesterol and triglycerides in lipoprotein fraction were lower (fig. 4k, l). After 8 weeks of HFHS feeding, Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were performed when there was no significant difference in body weight between the two groups of mice. L-Usp20^-/-The mouse is inA significant improvement in glucose clearance and insulin sensitivity was shown (fig. 4m, n). The results show that L-Usp20^-/-The mice have strong improvement effect on diet-induced obesity (DIO), fatty liver and insulin resistance.

We are dealing with Usp20^KI/KIMice were similarly analyzed. Usp20 after 14 weeks of HFHS feeding^KI/KIThere was no significant difference in body weight, food intake, fat mass, lean mass and liver weight from WT mice, although there was a tendency for both body weight and liver weight to decrease (fig. 15 c-g). More importantly, by GTT and ITT experiments, Usp20^KI/KIMice showed significantly improved glucose clearance and insulin sensitivity (fig. 15h, i). These results show that L-Usp20^-/-And Usp20^KI/KIMice all showed improved metabolic profiles on the HFHS diet.

In view of WT and L-Usp20^-/-Mice ingested similar amounts of food but varied in body weight, and energy expenditure was subsequently examined 10 weeks after HFHS feeding. L-Usp20^-/-Mice showed significant increases in oxygen consumption both at night and during the day (FIG. 4o, p) but WT and L-Usp20^-/-There were no differences in Respiratory Entropy (RER) and physical activity between mice (fig. 15j, k). By IR photographic analysis, L-Usp20^-/-The body temperature of the mice was about 1 degree higher than that of the WT mice (fig. 4q, r). L-Usp20^-/-Mice showed higher rectal temperatures at 4 ℃ (fig. 4 s). Indicating that USP20 deficiency promotes thermogenesis in mice.

Studies have shown that succinic acid is an intermediate in the tricarboxylic acid (TCA) cycle and can activate thermogenesis. We have detected L-Usp20^-/-Changes in TCA cycle metabolic intermediates in mouse liver. L-Usp20^-/-The succinic acid concentration of mouse liver was about 2 times that of WT mouse. Fumaric acid was also slightly increased, while other TCA intermediates were in L-Usp20^-/-Maintained or even reduced in mice (FIG. 4t, u) and L-Usp20^-/-The level of succinic acid in the serum of the mice was increased by one-fold (fig. 4 v).

At the same time, we also detected L-Usp20^-/-Several known thermogenic regulators in mouse serum, including epinephrineNorepinephrine, thyroxine, triiodothyroxine, secretin, thyroid stimulating hormone and FGF 21. Their levels in serum were not found to be altered (FIG. 151-r). L-Usp20^-/-There was also no difference with the serum ALT and AST of WT mice (fig. 15s, t), indicating no liver damage. In conclusion, knockout of Usp20 in the liver reduces lipid biosynthesis and increases succinate levels leading to thermogenesis and L-Usp20^-/-Mice were resistant to diet-induced obesity (DIO), showing lower lipid levels and higher insulin sensitivity.

Example 19 USP20 inhibitors improve metabolic syndrome

L-Usp20^-/-The metabolic improvement in mice suggests that USP20 may be a therapeutic target for obesity and related metabolic syndrome. GSK2643943A is a potent specific inhibitor of USP20 and can be used as a tool to study the pharmacological inhibitory effects of USP20 in vivo (fig. 16a, b)³⁸. WT mice fed normal diet were orally administered GSK2643943A (30 mg/kg/day) daily for 13 consecutive days. Mice were then starved-refed. GSK2643943A clearly inhibited the food-induced increase in HMGCR protein, however, this did not alter the USP20 phosphorylation modification and total protein level, nor the expression level of the FASN protein (fig. 5b), the table name USP20 inhibitor specifically affected HMGCR. USP20 inhibitor significantly reduced serum levels of TC and TG both under starvation and refeeding conditions (FIG. 5c, d, FIG. 14), which is in contrast to L-Usp20^-/-And Usp20^KI/KIThe findings in mice were consistent (fig. 4 and 14). In addition, GTT results showed a significant increase in glucose clearance in GSK2643943A treated mice (figure 16 c). AST and ALT results indicate that drug treatment did not result in liver damage (FIGS. 16 d-e). GSK2643943A significantly increased the energy expenditure of mice during day and night (fig. 5e, f). Consistent with the previous results, succinic acid levels in serum and liver were significantly increased in mice treated with GSK2643943A (fig. 5g, h).

The improved glycolipid metabolism by inhibition of USP20 prompted us to further analyze whether GSK2643943A could be used to treat diet-induced obesity (DIO) and obesity-related diseases such as insulin resistance, hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). WT mice were fed with a high-sugar, high-fat diet for 15 weeks to induce obesity, and then orally gavage mice with GSK2643943A for 2 weeks (fig. 5 i). The results showed that drug treatment had no effect on mouse food intake (fig. 16f), but caused a significant weight loss in mice, and body fat analysis showed that weight loss was mainly due to weight loss in adipose and liver tissues (fig. 16 i). Drug treatment had no effect on the weight of brown adipose tissue, kidney and heart (fig. 16 j-1). GSK2643943A significantly reduced lipid levels in liver and serum (fig. 5k-n) and adipocyte size (fig. 5o, p). HMGCR protein levels were significantly reduced in the liver of mice treated with GSK2643943A (fig. 16 m). Total oxygen consumption was significantly higher in GSK2643943A treated mice than in control mice (fig. 16n, o). At the same time, GSK2643943A greatly increased mouse glucose clearance (fig. 5 q). Finally, the serum AST, ALT and creatinine concentrations were similar in the control and GSK2643943A treated mice (FIG. 16p-r), indicating no liver and kidney toxicity. GSK2643943A also did not alter serum TNF α levels, the expression of macrophage markers (CD68, F4/80 and Arg-1) and inflammatory markers (TNF α and IL6) (fig. 16s, t). Taken together, these results indicate that the dosage of USP20 inhibitor we used is safe and a promising therapeutic candidate for the treatment of metabolic diseases such as obesity, cardiovascular and cerebrovascular diseases, hyperlipidemia, non-alcoholic fatty liver disease, diabetes and the like.

Discussion of the related Art

Cholesterol synthesis is tightly regulated by SREBP-regulated transcriptional levels and gp 78-mediated post-translational levels of HMGCR degradation. Lanosterol, an intermediate product of cholesterol synthesis, promotes the degradation of HMGCR to ensure a relatively stable cholesterol concentration. Mammals have evolved multiple mechanisms that can efficiently store energy by increasing glycogen and lipid synthesis after feeding³⁹. The biosynthesis of cholesterol also increases after eating. Here, we determined that USP20 is a key factor in responding to an increase in hepatic cholesterol biosynthesis after eating. Feeding increases the levels of insulin and glucose in the blood, thereby promoting mTOR-mediated phosphorylation of USP 20. This results in binding of USP20 to gp78, which is stableThe HMGCR is determined, the biosynthesis of cholesterol is increased, and the cholesterol can be transported to other tissues through circulating system lipoprotein so as to meet the needs of the organism. In addition, since a sterol intermediate is also produced in the mevalonate pathway as an endogenous LXR ligand, increased HMGCR protein can activate LXR, thereby increasing fatty acid synthesis.

mTOR is an organizer of vital nutrient sensors and energy storage after eating. We found that USP20 is regulated by the mTOR signaling pathway to promote cholesterol synthesis. Knockout or compound inhibition of USP20 may arrest this nutrient saving mechanism and provide various positive benefits to metabolism and health. First, cholesterol biosynthesis is turned off due to the reduction of HMGCR protein. Second, because endogenous LXR ligands cannot be produced, SREBP1c levels and fatty acid synthesis gene expression are reduced, and fatty acid synthesis is also reduced. Third, when USP20 is inhibited, succinic acid can be increased by increasing the TCA intermediate³⁷Thereby increasing energy consumption. We found that succinic acid concentration increased after feeding, while USP20 was inhibited, its concentration further increased (FIGS. 4t-v, FIGS. 5 g-h). Although the mechanism is not clear, it is known that Succinate Dehydrogenase (SDH) is modified by acetylation to inhibit its activity⁴⁰. Feeding may reduce SIRT3 deacetylase by increasing the concentration of acetyl-CoA⁴¹To facilitate SDH acetylation. Inhibition of USP20 may further increase SDH acetylation, thereby increasing succinic acid levels.

In conclusion, the invention provides a new application of USP20 as a therapeutic target for reducing cholesterol synthesis caused by eating and improving metabolic syndrome by researching the function of deubiquitinase USP20 in cholesterol synthesis regulation.

The sequence is as follows:

SEQ ID NO.1

MGDSRDLCPHLDSIGEVTKEDLLLKSKGTCQSCGVTGPNLWACLQVACPYVGCGESFADHSTIHAQAKK HNLTVNLTTFRLWCYACEKEVFLEQRLAAPLLGSSSKFSEQDSPPPSHPLKAVPIAVADEGESESEDDDLK PRGLTGMKNLGNSCYMNAALQALSNCPPLTQFFLECGGLVRTDKKPALCKSYQKLVSEVWHKKRPSYV VPTSLSHGIKLVNPMFRGYAQQDTQEFLRCLMDQLHEELKEPVVATVALTEARDSDSSDTDEKREGDRS PSEDEFLSCDSSSDRGEGDGQGRGGGSSQAETELLIPDEAGRAISEKERMKDRKFSWGQQRTNSEQVDED ADVDTAMAALDDQPAEAQPPSPRSSSPCRTPEPDNDAHLRSSSRPCSPVHHHEGHAKLSSSPPRASPVRM APSYVLKKAQVLSAGSRRRKEQRYRSVISDIFDGSILSLVQCLTCDRVSTTVETFQDLSLPIPGKEDLAKL HSAIYQNVPAKPGACGDSYAAQGWLAFIVEYIRRFVVSCTPSWFWGPVVTLEDCLAAFFAADELKGDN MYSCERCKKLRNGVKYCKVLRLPEILCIHLKRFRHEVMYSFKINSHVSFPLEGLDLRPFLAKECTSQITTY DLLSVICHHGTAGSGHYIAYCQNVINGQWYEFDDQYVTEVHETVVQNAEGYVLFYRKSSEEAMRERQQ VVSLAAMREPSLLRFYVSREWLNKFNTFAEPGPITNQTFLCSHGGIPPHKYHYIDDLVVILPQNVWEHLY NRFGGGPAVNHLYVCSICQVEIEALAKRRRIEIDTFIKLNKAFQAEESPGVIYCISMQWFREWEAFVKGKD NEPPGPIDNSRIAQVKGSGHVQLKQGADYGQISEETWTYLNSLYGGGPEIAIRQSVAQPLGPENLHGEQKI EAETRAV

SEQ ID NO.2

ATGGGGGACTCCAGGGACCTTTGCCCTCACCTTGACTCCATAGGAGAGGTGACCAAAGAGGACTTG CTGCTCAAATCTAAGGGAACCTGTCAGTCGTGTGGGGTCACCGGACCAAACCTATGGGCCTGTCTGC AGGTTGCCTGCCCCTATGTTGGCTGCGGAGAATCCTTTGCTGACCACAGCACCATTCATGCACAGGC AAAAAAGCACAACTTGACCGTGAACCTGACCACGTTCCGACTGTGGTGTTACGCCTGTGAGAAGGA GGTATTCCTGGAGCAGCGGCTGGCAGCCCCTCTGCTGGGCTCCTCTTCCAAGTTCTCTGAACAGGAC TCCCCGCCACCCTCCCACCCTCTGAAAGCTGTTCCTATTGCTGTGGCTGATGAAGGAGAGTCTGAGT CAGAGGACGATGACCTGAAACCTCGAGGCCTCACGGGCATGAAGAACCTCGGGAACTCCTGCTACA TGAACGCTGCCCTGCAGGCCCTGTCCAATTGCCCGCCGCTGACTCAGTTCTTCTTGGAGTGTGGCGG CCTGGTGCGCACAGATAAGAAGCCAGCCCTGTGCAAGAGCTACCAGAAGCTGGTCTCTGAGGTCTG GCATAAGAAACGGCCAAGCTACGTGGTCCCCACCAGTCTGTCTCATGGGATCAAGTTGGTCAACCCA ATGTTCCGAGGCTATGCCCAGCAGGACACCCAAGAGTTCCTTCGCTGCCTGATGGACCAGCTGCACG AGGAGCTCAAGGAGCCGGTGGTGGCCACGGTGGCGCTGACGGAGGCTCGGGACTCAGATTCGAGTG ACACGGATGAGAAACGGGAGGGTGACCGGAGCCCATCAGAAGATGAGTTCTTGTCCTGTGACTCGA GCAGTGACCGGGGTGAGGGTGACGGGCAGGGGCGTGGCGGGGGCAGCTCGCAGGCCGAGACGGAG CTGCTGATCCCAGATGAGGCGGGCCGAGCCATCTCTGAGAAGGAGCGGATGAAGGACCGCAAGTTC TCCTGGGGCCAGCAGCGTACAAACTCGGAGCAAGTGGACGAGGACGCTGATGTGGACACTGCCATG GCTGCCCTTGACGACCAGCCCGCGGAGGCCCAGCCCCCGTCACCACGGTCCTCCAGCCCCTGCCGGA CGCCAGAGCCGGACAATGATGCTCACCTACGCAGCTCCTCTCGCCCCTGCAGCCCCGTCCACCACCA CGAGGGCCATGCCAAGCTGTCTAGCAGCCCCCCTCGTGCAAGCCCCGTGAGGATGGCACCGTCGTA CGTGCTCAAGAAAGCCCAGGTATTGAGTGCTGGCAGCCGGAGGCGGAAGGAGCAGCGCTACCGCAG CGTCATCTCAGACATCTTTGACGGCTCCATTCTCAGCCTTGTGCAGTGTCTCACCTGTGACCGGGTAT CCACCACAGTGGAAACGTTCCAGGACTTATCACTGCCCATTCCTGGAAAGGAGGACCTGGCCAAGC TCCATTCAGCCATCTACCAGAATGTGCCGGCCAAGCCAGGCGCCTGTGGGGACAGCTATGCCGCCCA GGGCTGGCTGGCCTTCATTGTGGAGTACATCCGACGGTTTGTGGTATCCTGTACCCCCAGCTGGTTTT GGGGGCCTGTCGTCACCCTGGAAGACTGCCTTGCTGCCTTCTTTGCCGCTGATGAGTTAAAGGGTGA CAACATGTACAGCTGTGAGCGGTGTAAGAAGCTGCGGAACGGAGTGAAGTACTGCAAAGTCCTGCG GTTGCCCGAGATCCTGTGCATTCACCTAAAGCGCTTTCGGCACGAGGTGATGTACTCATTCAAGATC AACAGCCACGTCTCCTTCCCCCTCGAGGGGCTCGACCTGCGCCCCTTCCTTGCCAAGGAGTGCACAT CCCAGATCACCACCTACGACCTCCTCTCGGTCATCTGCCACCACGGCACGGCAGGCAGTGGGCACTA CATCGCCTACTGCCAGAACGTGATCAATGGGCAGTGGTACGAGTTTGATGACCAGTACGTCACAGA AGTCCACGAGACGGTGGTGCAGAACGCCGAGGGCTACGTACTCTTCTACAGGAAGAGCAGCGAGGA GGCCATGCGGGAGCGACAGCAGGTGGTGTCCCTGGCCGCCATGCGGGAGCCCAGCCTGCTGCGGTT CTACGTGTCCCGCGAGTGGCTCAACAAGTTCAACACCTTCGCGGAGCCAGGCCCCATCACCAACCAG ACCTTCCTCTGCTCCCACGGAGGCATCCCGCCCCACAAATACCACTACATCGACGACCTGGTGGTCA TCCTGCCCCAGAACGTCTGGGAGCACCTGTACAACAGATTCGGGGGTGGCCCCGCCGTGAACCACCT GTACGTGTGCTCCATCTGCCAGGTGGAGATCGAGGCACTGGCCAAGCGCAGGAGGATCGAGATCGA CACCTTCATCAAGTTGAACAAGGCCTTCCAGGCCGAGGAGTCGCCGGGCGTCATCTACTGCATCAGC ATGCAGTGGTTCCGGGAGTGGGAGGCGTTCGTCAAGGGGAAGGACAACGAGCCCCCCGGGCCCATT GACAACAGCAGGATTGCACAGGTCAAAGGAAGCGGCCATGTCCAGCTGAAGCAGGGAGCTGACTAC GGGCAGATTTCGGAGGAGACCTGGACCTACCTGAACAGCCTGTATGGAGGTGGCCCCGAGATTGCC ATCCGCCAGAGTGTGGCGCAGCCGCTGGGCCCAGAGAACCTGCACGGGGAGCAGAAGATCGAAGC CGAGACGCGGGCCGTGTGA

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SEQUENCE LISTING

<110> Wuhan university

<120> use of deubiquitinase USP20 for reducing cholesterol synthesis and improving metabolic syndrome

<130> WH1954-19P122709

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 914

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Asp Ser Arg Asp Leu Cys Pro His Leu Asp Ser Ile Gly Glu

1 5 10 15

Val Thr Lys Glu Asp Leu Leu Leu Lys Ser Lys Gly Thr Cys Gln Ser

20 25 30

Cys Gly Val Thr Gly Pro Asn Leu Trp Ala Cys Leu Gln Val Ala Cys

35 40 45

Pro Tyr Val Gly Cys Gly Glu Ser Phe Ala Asp His Ser Thr Ile His

50 55 60

Ala Gln Ala Lys Lys His Asn Leu Thr Val Asn Leu Thr Thr Phe Arg

65 70 75 80

Leu Trp Cys Tyr Ala Cys Glu Lys Glu Val Phe Leu Glu Gln Arg Leu

85 90 95

Ala Ala Pro Leu Leu Gly Ser Ser Ser Lys Phe Ser Glu Gln Asp Ser

100 105 110

Pro Pro Pro Ser His Pro Leu Lys Ala Val Pro Ile Ala Val Ala Asp

115 120 125

Glu Gly Glu Ser Glu Ser Glu Asp Asp Asp Leu Lys Pro Arg Gly Leu

130 135 140

Thr Gly Met Lys Asn Leu Gly Asn Ser Cys Tyr Met Asn Ala Ala Leu

145 150 155 160

Gln Ala Leu Ser Asn Cys Pro Pro Leu Thr Gln Phe Phe Leu Glu Cys

165 170 175

Gly Gly Leu Val Arg Thr Asp Lys Lys Pro Ala Leu Cys Lys Ser Tyr

180 185 190

Gln Lys Leu Val Ser Glu Val Trp His Lys Lys Arg Pro Ser Tyr Val

195 200 205

Val Pro Thr Ser Leu Ser His Gly Ile Lys Leu Val Asn Pro Met Phe

210 215 220

Arg Gly Tyr Ala Gln Gln Asp Thr Gln Glu Phe Leu Arg Cys Leu Met

225 230 235 240

Asp Gln Leu His Glu Glu Leu Lys Glu Pro Val Val Ala Thr Val Ala

245 250 255

Leu Thr Glu Ala Arg Asp Ser Asp Ser Ser Asp Thr Asp Glu Lys Arg

260 265 270

Glu Gly Asp Arg Ser Pro Ser Glu Asp Glu Phe Leu Ser Cys Asp Ser

275 280 285

Ser Ser Asp Arg Gly Glu Gly Asp Gly Gln Gly Arg Gly Gly Gly Ser

290 295 300

Ser Gln Ala Glu Thr Glu Leu Leu Ile Pro Asp Glu Ala Gly Arg Ala

305 310 315 320

Ile Ser Glu Lys Glu Arg Met Lys Asp Arg Lys Phe Ser Trp Gly Gln

325 330 335

Gln Arg Thr Asn Ser Glu Gln Val Asp Glu Asp Ala Asp Val Asp Thr

340 345 350

Ala Met Ala Ala Leu Asp Asp Gln Pro Ala Glu Ala Gln Pro Pro Ser

355 360 365

Pro Arg Ser Ser Ser Pro Cys Arg Thr Pro Glu Pro Asp Asn Asp Ala

370 375 380

His Leu Arg Ser Ser Ser Arg Pro Cys Ser Pro Val His His His Glu

385 390 395 400

Gly His Ala Lys Leu Ser Ser Ser Pro Pro Arg Ala Ser Pro Val Arg

405 410 415

Met Ala Pro Ser Tyr Val Leu Lys Lys Ala Gln Val Leu Ser Ala Gly

420 425 430

Ser Arg Arg Arg Lys Glu Gln Arg Tyr Arg Ser Val Ile Ser Asp Ile

435 440 445

Phe Asp Gly Ser Ile Leu Ser Leu Val Gln Cys Leu Thr Cys Asp Arg

450 455 460

Val Ser Thr Thr Val Glu Thr Phe Gln Asp Leu Ser Leu Pro Ile Pro

465 470 475 480

Gly Lys Glu Asp Leu Ala Lys Leu His Ser Ala Ile Tyr Gln Asn Val

485 490 495

Pro Ala Lys Pro Gly Ala Cys Gly Asp Ser Tyr Ala Ala Gln Gly Trp

500 505 510

Leu Ala Phe Ile Val Glu Tyr Ile Arg Arg Phe Val Val Ser Cys Thr

515 520 525

Pro Ser Trp Phe Trp Gly Pro Val Val Thr Leu Glu Asp Cys Leu Ala

530 535 540

Ala Phe Phe Ala Ala Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys

545 550 555 560

Glu Arg Cys Lys Lys Leu Arg Asn Gly Val Lys Tyr Cys Lys Val Leu

565 570 575

Arg Leu Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu

580 585 590

Val Met Tyr Ser Phe Lys Ile Asn Ser His Val Ser Phe Pro Leu Glu

595 600 605

Gly Leu Asp Leu Arg Pro Phe Leu Ala Lys Glu Cys Thr Ser Gln Ile

610 615 620

Thr Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Gly

625 630 635 640

Ser Gly His Tyr Ile Ala Tyr Cys Gln Asn Val Ile Asn Gly Gln Trp

645 650 655

Tyr Glu Phe Asp Asp Gln Tyr Val Thr Glu Val His Glu Thr Val Val

660 665 670

Gln Asn Ala Glu Gly Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu

675 680 685

Ala Met Arg Glu Arg Gln Gln Val Val Ser Leu Ala Ala Met Arg Glu

690 695 700

Pro Ser Leu Leu Arg Phe Tyr Val Ser Arg Glu Trp Leu Asn Lys Phe

705 710 715 720

Asn Thr Phe Ala Glu Pro Gly Pro Ile Thr Asn Gln Thr Phe Leu Cys

725 730 735

Ser His Gly Gly Ile Pro Pro His Lys Tyr His Tyr Ile Asp Asp Leu

740 745 750

Val Val Ile Leu Pro Gln Asn Val Trp Glu His Leu Tyr Asn Arg Phe

755 760 765

Gly Gly Gly Pro Ala Val Asn His Leu Tyr Val Cys Ser Ile Cys Gln

770 775 780

Val Glu Ile Glu Ala Leu Ala Lys Arg Arg Arg Ile Glu Ile Asp Thr

785 790 795 800

Phe Ile Lys Leu Asn Lys Ala Phe Gln Ala Glu Glu Ser Pro Gly Val

805 810 815

Ile Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ala Phe Val

820 825 830

Lys Gly Lys Asp Asn Glu Pro Pro Gly Pro Ile Asp Asn Ser Arg Ile

835 840 845

Ala Gln Val Lys Gly Ser Gly His Val Gln Leu Lys Gln Gly Ala Asp

850 855 860

Tyr Gly Gln Ile Ser Glu Glu Thr Trp Thr Tyr Leu Asn Ser Leu Tyr

865 870 875 880

Gly Gly Gly Pro Glu Ile Ala Ile Arg Gln Ser Val Ala Gln Pro Leu

885 890 895

Gly Pro Glu Asn Leu His Gly Glu Gln Lys Ile Glu Ala Glu Thr Arg

900 905 910

Ala Val

<210> 2

<211> 2745

<212> DNA

<213> Homo sapiens

<400> 2

atgggggact ccagggacct ttgccctcac cttgactcca taggagaggt gaccaaagag 60

gacttgctgc tcaaatctaa gggaacctgt cagtcgtgtg gggtcaccgg accaaaccta 120

tgggcctgtc tgcaggttgc ctgcccctat gttggctgcg gagaatcctt tgctgaccac 180

agcaccattc atgcacaggc aaaaaagcac aacttgaccg tgaacctgac cacgttccga 240

ctgtggtgtt acgcctgtga gaaggaggta ttcctggagc agcggctggc agcccctctg 300

ctgggctcct cttccaagtt ctctgaacag gactccccgc caccctccca ccctctgaaa 360

gctgttccta ttgctgtggc tgatgaagga gagtctgagt cagaggacga tgacctgaaa 420

cctcgaggcc tcacgggcat gaagaacctc gggaactcct gctacatgaa cgctgccctg 480

caggccctgt ccaattgccc gccgctgact cagttcttct tggagtgtgg cggcctggtg 540

cgcacagata agaagccagc cctgtgcaag agctaccaga agctggtctc tgaggtctgg 600

cataagaaac ggccaagcta cgtggtcccc accagtctgt ctcatgggat caagttggtc 660

aacccaatgt tccgaggcta tgcccagcag gacacccaag agttccttcg ctgcctgatg 720

gaccagctgc acgaggagct caaggagccg gtggtggcca cggtggcgct gacggaggct 780

cgggactcag attcgagtga cacggatgag aaacgggagg gtgaccggag cccatcagaa 840

gatgagttct tgtcctgtga ctcgagcagt gaccggggtg agggtgacgg gcaggggcgt 900

ggcgggggca gctcgcaggc cgagacggag ctgctgatcc cagatgaggc gggccgagcc 960

atctctgaga aggagcggat gaaggaccgc aagttctcct ggggccagca gcgtacaaac 1020

tcggagcaag tggacgagga cgctgatgtg gacactgcca tggctgccct tgacgaccag 1080

cccgcggagg cccagccccc gtcaccacgg tcctccagcc cctgccggac gccagagccg 1140

gacaatgatg ctcacctacg cagctcctct cgcccctgca gccccgtcca ccaccacgag 1200

ggccatgcca agctgtctag cagcccccct cgtgcaagcc ccgtgaggat ggcaccgtcg 1260

tacgtgctca agaaagccca ggtattgagt gctggcagcc ggaggcggaa ggagcagcgc 1320

taccgcagcg tcatctcaga catctttgac ggctccattc tcagccttgt gcagtgtctc 1380

acctgtgacc gggtatccac cacagtggaa acgttccagg acttatcact gcccattcct 1440

ggaaaggagg acctggccaa gctccattca gccatctacc agaatgtgcc ggccaagcca 1500

ggcgcctgtg gggacagcta tgccgcccag ggctggctgg ccttcattgt ggagtacatc 1560

cgacggtttg tggtatcctg tacccccagc tggttttggg ggcctgtcgt caccctggaa 1620

gactgccttg ctgccttctt tgccgctgat gagttaaagg gtgacaacat gtacagctgt 1680

gagcggtgta agaagctgcg gaacggagtg aagtactgca aagtcctgcg gttgcccgag 1740

atcctgtgca ttcacctaaa gcgctttcgg cacgaggtga tgtactcatt caagatcaac 1800

agccacgtct ccttccccct cgaggggctc gacctgcgcc ccttccttgc caaggagtgc 1860

acatcccaga tcaccaccta cgacctcctc tcggtcatct gccaccacgg cacggcaggc 1920

agtgggcact acatcgccta ctgccagaac gtgatcaatg ggcagtggta cgagtttgat 1980

gaccagtacg tcacagaagt ccacgagacg gtggtgcaga acgccgaggg ctacgtactc 2040

ttctacagga agagcagcga ggaggccatg cgggagcgac agcaggtggt gtccctggcc 2100

gccatgcggg agcccagcct gctgcggttc tacgtgtccc gcgagtggct caacaagttc 2160

aacaccttcg cggagccagg ccccatcacc aaccagacct tcctctgctc ccacggaggc 2220

atcccgcccc acaaatacca ctacatcgac gacctggtgg tcatcctgcc ccagaacgtc 2280

tgggagcacc tgtacaacag attcgggggt ggccccgccg tgaaccacct gtacgtgtgc 2340

tccatctgcc aggtggagat cgaggcactg gccaagcgca ggaggatcga gatcgacacc 2400

ttcatcaagt tgaacaaggc cttccaggcc gaggagtcgc cgggcgtcat ctactgcatc 2460

agcatgcagt ggttccggga gtgggaggcg ttcgtcaagg ggaaggacaa cgagcccccc 2520

gggcccattg acaacagcag gattgcacag gtcaaaggaa gcggccatgt ccagctgaag 2580

cagggagctg actacgggca gatttcggag gagacctgga cctacctgaa cagcctgtat 2640

ggaggtggcc ccgagattgc catccgccag agtgtggcgc agccgctggg cccagagaac 2700

ctgcacgggg agcagaagat cgaagccgag acgcgggccg tgtga 2745

Claims (8)

1. Use of an antagonist or inhibitor of the deubiquitinase USP20 protein or of a gene encoding it for the manufacture of a medicament and/or therapeutic agent for reducing cholesterol and fatty acid synthesis and ameliorating metabolic syndrome, said antagonist or inhibitor being selected from the group consisting of:

an agent that inhibits the enzymatic activity of USP 20;

an agent that reduces the expression level and phosphorylation level of USP20 protein;

substances that reduce or block USP20 binding to gp 78; and/or

A substance which reduces or blocks the expression of a gene encoding deubiquitinase USP 20.

2. The use of claim 1, wherein the antagonist or inhibitor is selected from the group consisting of: an antibody or fusion protein directed against deubiquitinase USP20 protein, or an antisense nucleic acid or siRNA directed against deubiquitinase USP20 gene.

3. The use of claim 2, wherein the antibody or fusion protein directed against deubiquitinase USP20 protein recognizes the Ser132 and/or Ser134 site of USP20 protein.

4. The use according to any one of claims 1 to 3, wherein the inhibitor of deubiquitinase USP20 protein is GSK 2643943A.

5. The use of claim 1, wherein the medicament and/or therapeutic agent is for: reducing serum and liver lipid levels, increasing insulin sensitivity, increasing energy expenditure.

6. The use according to claim 5, wherein the medicament and/or therapeutic agent is for lowering cholesterol and lowering triglycerides.

7. The use according to claim 1, wherein the medicament and/or therapeutic agent is for the treatment and/or prevention of: weight gain caused by diet, obesity, hyperlipidemia, cardiovascular and cerebrovascular diseases, non-alcoholic fatty liver disease, and diabetes.

8. Use of a deubiquitinase USP20 protein or a gene encoding the same, wherein the deubiquitinase USP20 protein or the gene encoding the same is used for screening drugs and/or therapeutic agents for diseases related to cholesterol synthesis and metabolic pathways.

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