WO2023006127A1 - Use of gp73 as treatment target and diagnostic marker for non-obese non-alcoholic fatty liver disease - Google Patents

Abstract

The present application relates to a use of GP73 as a treatment target and a diagnostic marker for non-obese non-alcoholic fatty liver disease. GP73 promotes the occurrence and acceleration progress of non-obese NAFLD, and the inhibition of GP73 GAP enzyme activity effectively blocks the non-obese NAFLD phenotype induced by GP73.

Description

Application of GP73 as a therapeutic target and diagnostic marker for non-obese nonalcoholic fatty liver disease

cross reference

This application claims the priority rights of the invention patent application submitted on July 28, 2021 with the application number 202110858189.8 and the title of the invention "GP73 as a therapeutic target and diagnostic marker for non-obese non-alcoholic fatty liver disease" , the entire contents of which are incorporated herein by reference.

technical field

This application relates to the field of biomedicine, in particular to an application of GP73 as a therapeutic target and diagnostic marker for non-obese non-alcoholic fatty liver disease.

Background technique

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with a prevalence of more than 25% in the global population, and can progress from simple fatty liver to nonalcoholic steatohepatitis (NASH), liver fibrosis, Cirrhosis, and hepatocellular carcinoma (HCC) (Brunt et al., 2015; Buzzetti et al., 2016). Although NAFLD is strongly associated with body mass index (BMI), diabetes, it also occurs in persons with normal BMI values and is referred to as lean or non-obese NAFLD (Eslam et al., 2020), with non-obese NAFLD accounting for a Alcoholic fatty liver disease affects more than 40% of the population. Patients with non-obese NAFLD are metabolically deranged and have a higher incidence of type 2 diabetes mellitus (T2DM), liver fibrosis, and severe liver disease compared with obese NAFLD. Liver disease-related mortality was approximately 2-fold higher in non-obese NAFLD populations than in obese NAFLD populations. Additionally, non-obese NAFLD occurs in children and adults of all ethnicities (Ye et al., 2020). Therefore, risk factors other than obesity may play a key role in the pathophysiology of NAFLD in non-obese individuals. In non-obese NAFLD patients, unique trends in gut microbial composition can be observed, yet the characteristics and risk factors of non-obese NAFLD have not been fully understood (Lee et al., 2020). Therefore, elucidating the specific mechanisms leading to the development and poorer prognosis of non-obese NAFLD patients will help to develop appropriate guidelines and interventions for the diagnosis and treatment of this liver disease.

The development of NAFLD involves a series of changes associated with lipid accumulation in hepatocytes, with abnormal accumulation of lipids and apolipoproteins arising from changes in lipoprotein production, conversion, catabolism, or export (Mato et al., 2019). Very low-density lipoprotein (VLDL) is a large particle complex (about 30-80nm), mainly composed of triglycerides (TGs), phospholipids and cholesterol (CHO), with apolipoprotein B (ApoB) on its lipid core . VLDL is synthesized and assembled in hepatocytes and then secreted into plasma for delivery to other organs through the circulation (Gibbons et al., 2004; Tiwari and Siddiqi, 2012). VLDL assembled and secreted in the liver plays an important role in controlling the levels of TGs and CHO in plasma, and defects in VLDL export affect lipid homeostasis and make hepatocytes more prone to reactive oxygen species (ROS), endoplasmic reticulum stress and autophagy (Fujita et al., 2009), but how VLDL is secreted from living cells remains an open question. It has been reported that changes in multiple pathways, including insulin response, fatty acid β-oxidation, lipid storage and transport, and autophagy, are closely related to the progression of NAFLD to liver cancer (Kutlu et al., 2018; Takakura et al. , 2019).

Rab GTPases are the largest family of guanosine triphosphate (GTP)-binding proteins involved in the regulation of multiple steps in vesicular trafficking in eukaryotic cells (Diekmann et al., 2011). Multiple Rab GTPases have been identified as regulators of hepatic lipoprotein secretion (Kiss and Nilsson, 2014; Li and Yu, 2016). Among them, Rab23 and Rab1b affect the secretion of ApoE, ApoB100 and albumin to different extents (Takacs et al., 2017). Rab cycles between an inactive state bound to guanosine diphosphate (GDP) and an active state bound to GTP, the conversion of Rab to GTP and to GDP is catalyzed by guanine nucleotide exchange factors, GTP hydrolysis It is carried out by GTPase activating protein (GAP) (Barr and Lambright, 2010). Some GAP enzymes have a conserved Tre2/Bub2/Cdc16 (TBC) domain to catalyze the hydrolysis of Rab GTP (Pan et al., 2006).

GP73 protein, also known as type II Golgi membrane protein (Golgi phosphoprotein 2, GOLPH2) and Golgi membrane protein 1 (Golgi membrane protein 1, GOLM1), is a type II Golgi transmembrane protein with an N-terminal transmembrane domain (Bachert et al., 2007). GP73 expression is low in normal liver tissue but increases in response to liver injury, viral infection, or ER stress (Hu et al., 2011; Kladney et al., 2002; Wei et al., 2019) . The normal physiological function of GP73 and the pathological changes caused by chronically elevated GP73 levels are still unclear.

The information disclosed in this background section is only intended to increase the understanding of the general background of the application, and should not be considered as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art.

Contents of the invention

purpose of invention

The purpose of this application is to provide the application of GP73 as a therapeutic target and diagnostic marker for non-obese non-alcoholic fatty liver disease; the inventors have proved through experiments that GP73 is a target for the treatment of non-obese NAFLD and a diagnostic Markers of non-obese NAFLD. In the examples of the present application, GP73 promotes the occurrence and accelerated progression of non-obese NAFLD, while inhibiting the GAP enzyme activity of GP73 effectively blocks the phenotype of non-obese NAFLD induced by GP73.

solution

For realizing the purpose of the application, the application provides the following technical solutions:

The first aspect of the present application is to provide a use of an inhibitor of GP73 in the preparation of a medicament for treating non-obese NAFLD.

In a specific embodiment, the inhibitor of GP73 is a GP73 mRNA inhibitor, a GP73 protein inhibitor and/or a GAP enzyme activity inhibitor of GP73.

Further preferably, the GP73 mRNA inhibitor is a small interfering RNA specific for GP73, preferably siRNA as shown in SEQ ID NO: 1: 5'-CCUGGUGGCCUGUGUUAUUTT-3';

Further preferably, the GP73 protein inhibitor is an anti-GP73 monoclonal antibody.

The second aspect of the present application is to provide a method for treating non-obese NAFLD, comprising: administering a therapeutically effective amount of a GP73 inhibitor to a subject in need.

In a specific embodiment, the GP73 inhibitor is a GP73 mRNA inhibitor, a GP73 protein inhibitor and/or a GAP enzyme activity inhibitor of GP73;

Further preferably, the GP73 mRNA inhibitor is a small interfering RNA specific for GP73, preferably siRNA as shown in SEQ ID NO: 1: 5'-CCUGGUGGCCUGUGUUAUUTT-3';

Further preferably, the GP73 protein inhibitor is an anti-GP73 monoclonal antibody.

The third aspect of the present application is to provide the use of the reagent for detecting GP73 in the preparation of a kit for diagnosing non-obese NAFLD.

In specific embodiments, the reagents for detecting GP73 include reagents for detecting its mRNA level, protein level or GAP enzyme activity level.

Further preferably, the reagents for detecting GP73 mRNA levels include detection primers or probes specific for GP73 gene.

Further preferably, the reagent for detecting the level of GP73 protein includes an antibody specific for GP73 protein.

Further preferably, the reagent for detecting the GAP enzyme activity level of GP73 includes a detection reagent for the phosphate released by GP73 hydrolyzing the substrate Rab protein through its GAP enzyme activity.

The fourth aspect of the present application is to provide the use of GP73 as a diagnostic marker for non-obese NAFLD.

The fifth aspect of the present application is to provide a method for diagnosing non-obese NAFLD, which includes: detecting the mRNA level of GP73, the protein level and/or the GAP enzyme activity level of GP73 in the subject, when compared to the normal control When the corresponding level of is elevated, the subject is diagnosed as obese NAFLD.

Beneficial effect

In this application, by revealing the common and specific characteristics of GP73-induced non-obese NAFLD, it is indicated that GP73 is a target for the treatment of non-obese NAFLD and also a marker for the diagnosis of non-obese NAFLD. In the examples of this application, the following results are shown: mice with long-term upregulation of GP73 in hepatocytes exhibit a metabolic phenotype, that is, they have almost all the characteristics of human non-obese NAFLD patients, and also have lipid metabolites in the liver GP73 promotes the occurrence and accelerated progression of non-obese NAFLD. Inhibition of the GAP enzyme activity of GP73 effectively blocked the non-obese NAFLD phenotype induced by GP73.

Description of drawings

One or more embodiments are exemplified by pictures in the accompanying drawings, and these exemplifications are not intended to limit the embodiments. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or better than other embodiments.

Figure 1A: Motif comparison of GP73 with Gyp1p, VirA, EspG and EspG2 with TBC domain.

Figure 1B: The ability of GP73 to catalyze the hydrolysis of 13 mammalian Rabs.

Figure 1C: Kinetic analysis of the hydrolytic activity of GP73 towards different concentrations of Rab23.

Fig. 1D: Effects of R248K and Q310A mutations at the key sites of GAP enzyme activity of GP73 on the GAP enzyme activity of GP73.

Fig. 2A: Flow chart of transfection of Flag-V, Flag-GP73 and Flag-GP73-RQ in Huh-7 cells, washing cells with PBS 24 hours later, and detection of supernatant secretion after 6 hours.

Figure 2B: Huh-7 cells were transfected with Flag-V, Flag-GP73 and Flag-GP73-RQ expression plasmids, and the expression level of GP73 in the cells was detected by immunoblotting after 24 and 48 hours.

Figure 2C: Detection of ApoB levels in the supernatant after transfection of Flag-V, Flag-GP73 and Flag-GP73-RQ expression plasmids in Huh-7 cells, respectively. ns means no statistical difference, ** means P<0.01.

Figure 2D: Detection of ApoB100 levels in the supernatant after transfection of Flag-V, Flag-GP73 and Flag-GP73-RQ expression plasmids in Huh-7 cells. ns means no statistical difference, ** means P<0.01.

Figure 2E-2G: ApoE (Figure 2E), albumin (Figure 2F) and ApoA1 (Figure 2G) in the supernatant after transfection of Flag-V, Flag-GP73 and Flag-GP73-RQ expression plasmids in Huh-7 cells level detection. ns means no statistical difference, * means P<0.05, ** means P<0.01, *** means P<0.001.

Figure 2H: Detection of the expression levels of GP73 and ApoB100 in the liver of mice injected with AAV-V or AAV-GP73.

Figure 2I: Detection of expression levels of triglycerides in plasma after mice injected with AAV-V, AAV-GP73 or AAV-GP73-RQ were treated with tyloxapol for 1, 2, and 3 hours. ns means no statistical difference, *** means P<0.001.

Figure 3A: HE staining and Oil Red O staining of livers of mice injected with AAV-V or AAV-GP73 fed with normal diet for 6 months.

3B-3D: Liver to body weight ratio (Fig. 3B), spleen (Fig. 3C), spleen to body weight ratio (Fig. 3D) of mice injected with AAV-V or AAV-GP73. * means P<0.05, ** means P<0.01

Figure 3E-3K: Triglyceride in liver (Figure 3E), cholesterol in liver (Figure 3F), triglyceride in plasma (Figure 3G), cholesterol in plasma (Figure 3H) in mice injected with AAV-V or AAV-GP73 ), alanine aminotransferase (ALT) (Fig. 3I), aspartate aminotransferase (AST) (Fig. 3J) levels, and mouse body weight (Fig. 3K). * means P<0.05, ** means P<0.01.

Figures 4A-4D: Fasting blood glucose of mice injected with AAV-V or AAV-GP73 after feeding for 6 weeks (Figure 4A), 15 weeks (Figure 4B), 18 weeks (Figure 4C), and 24 weeks (Figure 4D). * means P<0.05, ** means P<0.01.

Figures 4E-4G: Glucose tolerance test (GTT) results of mice injected with AAV-V or AAV-GP73 after feeding for 6 weeks (Figure 4E), 15 weeks (Figure 4F), and 18 weeks (Figure 4G). ns means no statistical difference, * means P<0.05, ** means P<0.01.

Figure 5A-5G: Plasma LDL (Figure 5A), TGs (Figure 5B), CHO (Figure 5C), ALT (Figure 5F) in mice injected with AAV-V, AAV-GP73 or AAV-GP73-RQ fed for 18 weeks , AST (Fig. 5G) levels and liver TGs (Fig. 5D) and CHO (Fig. 5E) levels. ns means no statistical difference, * means P<0.05, ** means P<0.01, *** means P<0.001.

Figures 5H-5K: Fasting blood glucose levels in mice injected with AAV-V, AAV-GP73 or AAV-GP73-RQ after feeding for 9 weeks (Figure 5H), 12 weeks (Figure 5I), and 16 weeks (Figure 5J), respectively, And the glucose tolerance test (GTT) results of mice injected with AAV-V, AAV-GP73 or AAV-GP73-RQ after 18 weeks of feeding (Fig. 5K). ns means no statistical difference, * means P<0.05, ** means P<0.01, *** means P<0.001.

Figure 6A: Screening of siRNA knockdown effects of different GP73s.

Fig. 6B: Detection of GP73 mRNA expression levels in mice fed with normal diet and high-fat diet after injection of control siRNA or siGP73. *** indicates P<0.001.

Figure 6C-6D: ALT (Figure 6C) and AST (Figure 6D) levels in plasma of mice injected with control siRNA or siGP73 fed with normal diet and high-fat diet **indicates P<0.01, ***indicates P<0.001 .

Figure 6E: Four weeks after mice were injected with control siRNA or siGP73, fasting blood glucose levels in mice fed normal chow (Reg) and high-fat and high-cholesterol chow (HFHCC) conditions. * indicates P<0.05.

Fig. 6F: Results of glucose tolerance test (GTT) in mice fed with normal diet (Reg) and high-fat and high-cholesterol diet (HFHCC) after injection of control siRNA or siGP7 for 34 weeks. * means P<0.05, ** means P<0.01.

Fig. 6G-6H: TG (Fig. 6G) and CHO (Fig. 6H) in liver tissue of mice injected with control siRNA or siGP73 under the conditions of normal diet (Reg) and high-fat and high-cholesterol diet (HFHCC) after 4 weeks )s level. * indicates P<0.05, ** indicates P<0.01.

Detailed ways

In order to make the purpose, technical solutions and advantages of the application clearer, the technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the application, rather than all implementations example. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of this application.

In addition, in order to better illustrate the present application, numerous specific details are given in the following specific implementation manners. It will be understood by those skilled in the art that the present application may be practiced without certain of the specific details. In some embodiments, materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail, so as to highlight the gist of the present application.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations thereof such as "includes" or "includes" and the like will be understood to include the stated elements or constituents, and not Other elements or other components are not excluded.

Experimental materials and methods

Reagent

Berberine (SML2577) and tyloxapol (T8761) were from Sigma-Aldrich (MO, USA).

Insulin (2018283062) was purchased from Novo Nordisk (Copenhagen, Denmark).

Glucose (20171108) was purchased from Sinopharm Chemical Reagent (Beijing) Co., Ltd.

Blood glucose meters (06656919032) and test strips (1072332990) were purchased from Roche (Basel, Switzerland).

Dulbecco's modified Eagle's medium (DMEM, high glucose, D5796) was purchased from Millipore (MA, USA).

Lipofectamine 2000 (11668027) was purchased from Invitrogen (Carlsbad, California, USA).

Diets containing HF (HD034a, 15% fat), HC (HD034c, 3% cholesterol) and HFHC (HD034b, 15% fat, 3% cholesterol) were purchased from Beijing Botai Hongda Biotechnology Co., Ltd. (Beijing, China), and Store according to manufacturer's recommendations.

PerfectStart ^TM Green qPCR SuperMix (AQ601) and

One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311) was purchased from TransGen Biotech (Beijing, China).

Mouse insulin ELISA kit (PI602) and BCA protein concentration assay kit (P0012) were purchased from Beyotime (Shanghai, China).

Mouse glycosylated hemoglobin kit (80420) was purchased from Crystal Chem (WA, USA).

Mouse biochemical AST (200218), ALT (191230), TG (200224) and CHO (200224) kits were purchased from Ruierda Biotechnology (Beijing, China).

Mouse ApoB (SEC003Mu), ApoB100 (PAA603Mu01), albumin (CEB028Mu), ApoE (SEA704Mu), ApoA1 (SEA519Mu), TG (CEB687Ge) and CHO (CEB701Ge) ELISA kits were purchased from Yunclone Company (Wuhan, China).

Mouse TGF-β (121702), IFN-γ (120062), IL-1β (1210122) and IL-6 (1210602) ELISA kits were purchased from Dakwi Biotechnology Co., Ltd. (Beijing, China).

Antibody

Anti-α-tubulin antibody (T6074, 1:5000 dilution) was purchased from Sigma-Aldrich Company. Anti-GP73 antibody (ab92612, 1:2000 dilution) was purchased from Abcam (Cambridge, UK). Anti-ApoB antibody (20578-1-AP, 1:1500 dilution) was purchased from Proteintech Group (Chicago, IL, USA). Anti-rabbit HRP-IgG (ZB-2301, 1:5000 dilution) and anti-mouse HRP-IgG (ZB-2305, 1:5000 dilution) were purchased from Zhongshan Jinqiao Company (Beijing, China).

plasmid

A mammalian expression plasmid encoding N-terminal Myc- and C-terminal Flag-tagged GP73 was constructed by inserting the corresponding PCR amplified fragment into the pcDNA3.1 (V79520, Invitrogen) vector. The expression plasmid pCDNA3.1-Flag-GP73-RQ was constructed by Fast Site-Directed Mutagenesis Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China). The sequences of all constructed plasmids were confirmed by sequence determination.

Cell Culture and Transfection

The Huh-7 cell line was purchased from the Japanese Collection of Research Bioresources and tested for mycoplasma without mycoplasma contamination. Huh-7 cells were cultured in DMEM at 37°C in a humidified environment of 5% CO2. All media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 1× non-essential amino acid solution and 10 mM sodium pyruvate. Transfection was performed using Lipofectamine 2000 (Invitrogen).

Recombinant protein and GAP enzyme activity analysis

Expression and purification in 293T cells The recombinant GP73 protein with 6×His tag was prepared and purified with B-PER 6×His Spin Purification Kit (Lonza, Inc.). The recombinant Rab protein with Flag tag was expressed in 293T cells, and was purified with pCDNA3.1-Flag Spin Purification Kit and commercial system (Lonza, Inc.). Protein concentration was performed using Amicon ultracentrifugal filter units (Millipore). Protein concentration was determined using the Bradford method (Bio-Rad) and protein purity was determined using Coomassie blue staining of SDS-PAGE gels.

GAP enzyme activity assay and enzymatic kinetic assay using EnzChek Phosphate Assay Kit (Invitrogen). 50μM Flag-Rabs and 20nM His-tagged GP73 protein were continuously reacted at 37°C for 60 minutes and the absorbance at 360nm was continuously detected by a multifunctional microplate reader (Tecan, Switzerland).

Western blot analysis

Tissues and cells for western blot analysis were prepared in the presence of 150mM NaCl, 50mM Tris-HCl pH 7.5, 0.1% w/v SDS, 0.5% w/v sodium na-deoxycholate, 1% v/v Nonidet P-40, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2.5mM sodium pyrophosphate, 1mM NaVO4, 10mM Lysis was performed in NaF and lysate purchased from protease inhibitors (Sigma). After lysis, centrifuge at 4°C and 13000×g for 15 minutes, and perform SDS-PAGE after centrifugation. The separated proteins were transferred to PVDF membranes for western blot analysis using primary antibodies. Digital images were analyzed with ImageJ. α-tubulin was used as an internal reference, and the levels of the control group were quantitatively analyzed.

secretion test

Cells were transfected with Flag-vector or Flag-GP73, and after 24 or 48 hours, the cells were washed with PBS, after which the cell contents were secreted into fresh medium. After 6 hours, the culture medium and cell lysate were collected, and the content of the cell content was determined by ELISA method.

The secretion rate refers to the secretion efficiency of the cells, defined as the ratio between the amount of cell contents secreted in the supernatant to the total contents (the amount of secreted cell contents plus the amount of intracellular contents).

Preparation of AAV vector expressing GP73 or GP73-RQ

AAV viruses encoding mouse GP73, GP73R248K and Q310A mutants were constructed (Shanghai Hanbio Co., Ltd.). The tail vein injection dose of mice was 3×10 ¹¹ vg.

experimental animals

Male C57BL/6N wild-type mice were purchased from Sibeifu Biotechnology Company (Beijing, China). All animal experiments were performed at the Animal Center of the Academy of Military Medical Sciences (Beijing, China) and were approved by the Animal Management Committee.

TG secretion detection

Mice were fasted overnight, and then intravenously injected tyloxapol (400 mg/kg body weight). Blood was collected at designated time points, and plasma TG content was determined according to the relevant kit manufacturer's instructions.

Quantitative detection of blood glucose, plasma metabolites, cytokines and hepatic lipids

After fasting for 6 h, blood was collected from the tail vein of the mice, and the fasting blood glucose of the mice was measured with a blood glucose meter (Roche). Random blood glucose levels in mice were measured at 9 am. For blood glucose levels greater than 630 mg/dL (the upper detection limit of the blood glucose meter), record the value as 630 mg/dL.

Plasma HbA1c, LDL, TG, CHO, ALT, AST and cytokine levels were detected according to relevant kit instructions. For the determination of liver lipids, approximately 100 mg of liver tissue homogenate was extracted in methanol for 12 hours in a solvent of chloroform:methanol (2:1v/v) to obtain lipids, and then used Mouse TG and CHO ELISA kits were used for quantitative detection.

Histological analysis

Mouse liver tissue was fixed with formalin fixative, further embedded in paraffin block, and then sectioned (5 μm). Paraffin sections were stained with hematoxylin, eosin (HE) and oil red O (ORO) solutions for histological analysis. NAFLD is graded by the NAS semi-quantitative scoring system. NAS<3 points can exclude NASH, and NAS>4 points can diagnose non-alcoholic steatohepatitis (NASH). Non-alcoholic simple fatty liver (NAFLD) is defined as those without intralobular inflammation, ballooning and fibrosis but with >33% hepatic steatosis, and those with less than this degree of steatosis are only called hepatic steatosis.

metabolic test

Insulin and glucose tolerance test: After the mice were fasted for 6 hours, the fasting glucose content was measured by venous blood collected by the tail-cut method, and then glucose (1.5 g/kg body weight) or human insulin (0.75 U/kg body weight) was injected through the tail vein. Blood glucose levels were determined by taking blood from the tail vein at 0, 30, 60, 90 and 120 minutes after injection.

Statistical Analysis

Data are presented as mean ± SEM or median (interquartile range). Differences between two independent samples were evaluated with a two-tailed test. Differences among multiple samples were analyzed using one-way or two-way analysis of variance. Significance values were set as follows: ns (not significant), P>0.05; *P<0.05; **, P<0.01; and ***, P<0.001. Statistical analysis was performed using GraphPad Prism 8.0.

Example 1, GP73 has TBC-domain GAP enzyme activity that inhibits VLDL secretion

Studies have shown that GTP hydrolysis among Rab GTPases is the most well-known GAP with a cognate catalytic Tre2/Bub2/Cdc16 (TBC) domain to catalyze Rab GTP hydrolysis (Pan et al., 2006), which has a conserved arginine Finger domains and unique catalytic glutamine residues ("glutamine fingers"). We analyzed the amino acid sequence of GP73, compared the amino acid sequence near R248 and Q310 in GP73 with the equivalent residues in Gyp1p, VirA, EspG and EspG2 with two-finger catalytic motifs in the TBC domain, and found that in GP73 amino acid The sequence has a conserved TBC GAP domain, which contains a unique glutamine finger domain in addition to the arginine finger motif (VxxDxxR) (Fig. 1A).

Next, we investigated the ability of the recombinant GP73 protein to hydrolyze Rab GTPases. We measured the catalytic efficiency (Kcat/Km) of GP73 against 13 mammalian Rab GTPases. Among the 13 tested mammalian Rab GTPases, GP73 had the highest hydrolytic activity on Rab23 (Fig. 1B).

We used different concentrations of Rab23-GTP and the GAP enzyme activity of GP73 for Rab23 to conduct kinetic analysis, and the catalytic efficiency of GP73 for Rab23 was about 2.01×10 ⁵ M ^-1 S ^-1 (Fig. 1C).

Next, we investigated the GAP enzyme activity of the R248K Q310A mutant of GP73 (GP73-RQ). The R248K Q310A mutant of GP73 lost the ability to catalyze the GTP hydrolytic activity in Rab23 (Fig. 1D). Thus, GP73 is a GAP enzyme with R248 and Q310 active enzyme sites.

Example 2, GP73 inhibits the secretion of ApoB through its GAP enzyme activity

Rab GTPase is involved in the regulation of intracellular vesicle transport and lipoprotein secretion, so we speculate that the GAP enzyme activity of GP73 may affect lipoprotein secretion in hepatocytes. To test this hypothesis, we first investigated the involvement of GP73 in hepatic substance secretion.

The Huh-7 cells were transfected with Flag-V, Flag-GP73 or Flag-GP73-RQ respectively, and the cells were washed with PBS 1-2 days after transfection, and the Huh-7 cells were cultured in fresh medium for 6 hours. The secretion rate was detected by ELISA method. The secretion rate was calculated as the ratio between the secretion content and the cell secretion content (secretion plus total cell secretion). The expression of GP73 protein in Huh-7 cells transfected with Flag-V, Flag-GP73 or Flag-GP73-RQ was detected, using α-tubulin as an internal reference (Fig. 2A-B). We found that wild-type GP73 significantly decreased the secretion of ApoB and ApoB100, while GP73-RQ was unable to affect the secretion of ApoB and ApoB100 (FIG. 2C-FIG. 2D). Notably, GP73 promoted the secretion of ApoE and albumin (Fig. 2E-Fig. 2F), but did not affect the secretion of ApoA1 (Fig. 2G). GP73-RQ showed the same activity in the secretion of albumin, ApoE and ApoAl (Fig. 2E, Fig. 2F, Fig. 2G). These results suggest that GP73's effect on the secretion of hepatic lipoproteins ApoB and ApoB100 is dependent on the GTP hydrolysis activity of GP73.

We further examined the regulatory effect of GP73 on lipoprotein secretion in vivo. To this end, 8-week-old C57BL/6 mice were injected with hepatocyte-specific adeno-associated virus (AAV) expressing empty vector (AAV-V) and GP73 (AAV-GP73), respectively, through the tail vein. liver. About three weeks after the injection of AAV virus, the expression of GP73 in the liver of mice infected with AAV-GP73 was increased by about 3 times (Fig. 2H). Western blot analysis showed that ApoB100 expression was significantly increased in the liver of AAV-GP73 mice (Fig. 2H). After the mice injected with AAV-V, mice injected with AAV-GP73 or mice injected with AAV-GP73-RQ were fasted for 4 hours, they were intravenously infused with tyloxapol (400 mg/kg), and the concentration of TG in the serum of the mice was detected. We observed a significant decrease in plasma TG levels in AAV-GP73-injected mice 1, 2, and 3 h after injection of tyloxapol, indicating a significant decrease in newly secreted VLDL (Fig. 2I). In contrast, hepatic expression of the AAV-GP73-RQ mutant did not affect VLDL secretion (Fig. 2I). Taken together, these data suggest that GP73 has an inhibitory effect on VLDL secretion that is dependent on its GAPase activity.

Example 3: Chronic Elevation of Hepatic GP73 Can Cause Non-Obese NAFLD

Since impaired VLDL secretion is closely associated with dyslipidemia, we further assessed the metabolic profile of chronic GP73 abnormal expression in hepatocytes.

AAV-V mice and AAV-GP73 mice were fed a normal diet for 24 weeks, after which liver tissues were collected. Compared with control mice, after the high expression of GP73 in the liver, the liver was slightly enlarged (Fig. 3A), and the ratio of liver to body weight and spleen to body weight were also significantly increased (Fig. 3B-D). Cell swelling and massive lipid accumulation (Fig. 3A). Liver TG and CHO levels also increased in mice with high liver GP73 expression (Fig. 3E-F). Importantly, serum total TG and CHO levels were significantly reduced in mice with high hepatic GP73 expression (Figures 3G and 3H), possibly as a result of impaired hepatic VLDL secretion. As indicators of liver damage, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were also significantly increased in AAV-GP73-infected mice (Fig. 3I–J). Mice with high hepatic GP73 expression exhibited reduced body weight compared to control mice (Fig. 3K). Thus, mice with chronic high hepatocyte GP73 expression display a non-obese NAFLD phenotype.

We next investigated whether lipotoxicity caused by chronic GP73 overexpression was associated with abnormal glucose metabolism. Six weeks after AAV injection in mice, there was no statistical difference in fasting blood glucose levels between mice with high liver GP73 expression and control mice (Fig. 4A). However, after week 15 after AAV injection, mice with high levels of hepatic GP73 had significantly higher fasting blood glucose levels compared to control mice (Fig. 4B), and this difference persisted until week 24 (Fig. 4C and 4D). . At week 18 after AAV-GP73 injection, mice with high liver GP73 expression developed severe glucose intolerance (Figures 4E, 4F, and 4G). We therefore conclude that chronically elevated levels of GP73 in hepatocytes cause non-obese NAFLD.

Example 4: GP73's induction of non-obese NAFLD is highly dependent on its GAP enzyme activity

We then assessed whether non-obese NAFLD induced by chronic increases in hepatocyte GP73 is primarily dependent on the GTPase activity of GP73. AAV-V, AAV-GP73 or AAV-GP73-RQ were injected tail vein into 8-week-old C57BL/6 mice. After 18 weeks of regular diet, the plasma LDL, TGs, CHO, ALT, AST levels and liver TGs and CHO levels of mice injected with AAV-V, AAV-GP73 and AAV-GP73-RQ were detected. In mice with high expression of GP73 in the liver, it was observed that plasma low-density lipoprotein (LDL) level (Fig. 5A), TG level (Fig. 5B) and CHO level (Fig. ) and CHO (Fig. 5E) levels were significantly increased. Plasma and liver lipid levels were unchanged in mice injected with AAV-GP73-RQ (Fig. 5A-E). Notably, mice injected with GP73-RQ did not exhibit liver damage (Fig. 5F-G).

To determine whether the GAP enzyme activity of GP73 regulates blood glucose, we performed a time-dependent assay of fasting blood glucose levels. We noticed a significant increase in fasting blood glucose levels in mice expressing WT GP73, whereas mice expressing GP73-RQ did not develop hyperglycemia throughout the test period (Fig. 5H–J). Eighteen weeks after injection, AAV-GP73-injected mice had impaired glucose tolerance, and GP73-RQ-injected mice exhibited normal glucose uptake (Fig. 5K). Thus, the GAP enzyme activity of GP73 is essential for its role in metabolism.

Example 5: GP73 blockade improves systemic metabolism in diet-induced non-obese NAFLD mice

To further explore whether inhibition of hepatic GP73 could improve metabolic disorders, we delivered siRNA of GP73 to the liver of mice. After screening 9 potential siRNA sequences, siGP73-1 (whose sequence is shown in SEQ ID NO: 1 as: 5′-CCUGGUGGCCUGUGUUAUUTT-3′) was identified, which produced better GP73 knockdown in vitro effect (as shown in Figure 6A). Next, we investigated the effect of sustained GP73 expression suppression on diet-induced lipid and glucose homeostasis in non-obese NAFLD mice by feeding 20-week-old C57BL/6 mice with HFHCC for 4 weeks. Then, inject the above-mentioned siGP73-1 or messy small interfering RNA control (corresponding to sicontrol in Fig. 6 , its sequence is as shown in SEQ ID NO: 2, being: 5'-AUCACACCAACACAGGUCCTT-3' to mice twice a week ). The results showed that after 4 weeks of siGP73-1 administration, GP73 mRNA was found to be down-regulated by about 50% in the liver (as shown in Figure 6B); and, siGP73 administration alleviated liver damage (as shown in Figure 6C-D), And resulted in a significant decrease in blood glucose levels (as shown in Figure 6E). Notably, knockdown of hepatic GP73 also improved insulin sensitivity in the intervention protocol (as shown in Figure 6F). Hepatocyte TG and CHO levels in the siGP73-treated group were lower than those in the control group (as shown in Figure 6G-H).

Taken together, these results suggest that GP73 blockade can improve systemic metabolic disturbances.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, rather than limiting them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present application.

Industrial Applicability

This application provides a use of GP73 as a therapeutic target and diagnostic marker for non-obese non-alcoholic fatty liver disease, which provides a valuable and promising candidate for the clinical treatment and diagnosis of non-obese non-alcoholic fatty liver disease Drugs and diagnostic markers.

Claims (9)

1. Use of an inhibitor of GP73 in the preparation of a medicament for treating non-obese NAFLD.
2. purposes according to claim 1, wherein, the inhibitor of described GP73 is the GAP enzyme activity inhibitor of GP73 mRNA inhibitor, GP73 protein inhibitor and/or GP73.
3. purposes according to claim 2, wherein, the mRNA inhibitor of described GP73 is the small interfering RNA specific for GP73, is preferably siRNA as shown in SEQ ID NO:1: 5 '-CCUGGUGGCCUGUGUUAUUTT-3';

   Preferably, the GP73 protein inhibitor is an anti-GP73 monoclonal antibody.
4. A method of treating non-obese NAFLD comprising: administering a therapeutically effective amount of an inhibitor of GP73 to a subject in need thereof.
5. The method according to claim 4, wherein the inhibitor of GP73 is a GAP enzyme activity inhibitor of GP73 mRNA inhibitor, GP73 protein inhibitor and/or GP73;

   Preferably, the mRNA inhibitor of GP73 is a small interfering RNA specific for GP73, preferably siRNA as shown in SEQ ID NO: 1: 5'-CCUGGUGGCCUGUGUUAUUTT-3';

   Preferably, the GP73 protein inhibitor is a GP73 monoclonal antibody.
6. Use of the reagent for detecting GP73 in the preparation of a kit for diagnosing non-obese NAFLD.
7. The use according to claim 6, wherein the reagent for detecting GP73 comprises a reagent for detecting its mRNA level, protein level or GAP enzyme activity level.
8. purposes according to claim 7, wherein, the reagent for detecting GP73 mRNA level comprises the detection primer or probe specific for GP73 gene;

   Preferably, the reagents for detecting the level of GP73 protein include antibodies specific for GP73 protein;

   Preferably, the reagent for detecting the level of GAP enzyme activity of GP73 includes a detection reagent for phosphate released by GP73 hydrolyzing the substrate Rab protein through its GAP enzyme activity.
9. A method for diagnosing non-obese NAFLD, comprising: detecting the mRNA level of GP73, the protein level and/or the GAP enzyme activity level of GP73 in a test subject, and when it increases relative to the corresponding level of a normal control, the The subjects were diagnosed with non-obese NAFLD.

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