Application of palmitoylation inhibitor in treating fatty liver disease related to metabolic dysfunction by inhibiting ACLY palmitoylation

Abstract

The invention relates to an application of a palmitoylation inhibitor in treating fatty liver diseases related to metabolic dysfunction by inhibiting ACLY palmitoylation. And the palmitoylation inhibitor is 2BP (2, 4-dibutyl phthalate). The fatty liver disease is a fatty liver disease related to metabolic dysfunction. The 2BP reduces the enzymatic activity of ACLY and the expression of lipid synthesis related protein by inhibiting palmitoylation of ACLY, thereby reducing lipid deposition. The invention provides application of 2BP in treating fatty liver diseases, and provides a new target of ACLY palmitoylation for treating fatty liver diseases related to metabolic dysfunction.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of a palmitoylation inhibitor, 2-bromopalmitic acid (2BP), in treating fatty liver disease associated with metabolic dysfunction by reducing the palmitoylation modification level of ATP-citrate lyase (ACLY). Background Art

[0002] ATP-citrate lyase (ACLY) is a key enzyme that plays a pivotal role in the cellular metabolic network. Its main function is to catalyze the conversion of mitochondrial citrate to cytoplasmic acetyl-CoA. The enzyme provides energy by hydrolyzing ATP, cleaving citrate into oxaloacetate and acetyl-CoA. Acetyl-CoA is an essential precursor for de novo fatty acid synthesis. This reaction establishes a core molecular bridge between glucose metabolism and lipid synthesis. Therefore, as a core converter connecting carbohydrate metabolism and anabolism, ACLY coordinates key biological processes such as cell proliferation, lipid homeostasis, and epigenetic regulation, playing an irreplaceable strategic role in physiological metabolic balance and the occurrence and development of diseases.

[0003] Palmitoylation is a dynamic, reversible, post-translational lipid modification of proteins, catalyzed by palmitoyltransferases, which covalently attach a hexadecyl palmitoyl group to a cysteine residue of a target protein via a thioester bond. This modification profoundly regulates protein subcellular localization, membrane domain partitioning, protein stability, conformational changes, and the assembly and dissociation of signaling complexes by enhancing protein hydrophobicity. 2-Bromopalmitate (2BP), a classic broad-spectrum inhibitor of palmitoylation, has a bromine atom substitution in its molecule that enables it to competitively mimic palmitoyl-CoA substrates, globally blocking the palmitoylation cycle. Palmitoylation plays a crucial role in the progression of various diseases.

[0004] Currently, there are no reports on the use of palmitoylation modification of palmitoylation inhibitors 2BP and ACLY in the treatment of metabolic dysfunction-related fatty liver disease. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide an application of a palmitoylation inhibitor 2BP in treating fatty liver disease associated with metabolic dysfunction by inhibiting ACLY palmitoylation modification.

[0006] In a first aspect, the present invention provides use of a palmitoylation inhibitor in treating fatty liver disease associated with metabolic dysfunction.

[0007] As a preferred example, the palmitoylation inhibitor is 2BP.

[0008] As another preferred example, the animal model of fatty liver disease refers to mice fed with a high-fat diet for 16 / 20 / 24 weeks.

[0009] As another preferred example, the 2BP inhibits the palmitoylation of ACLY.

[0010] As another preferred example, the reduction of ACLY palmitoylation will lead to decreased enzyme activity and reduced lipid synthesis.

[0011] In this study, we detected an overall increase in protein palmitoylation in the liver tissue of mice fed a high-fat diet using acyl-biotin exchange. Furthermore, we screened for palmitoylation of ACLY using acyl-resin-assisted capture coupled with mass spectrometry. Palmitic acid (PA) and the palmitoylation agonist PalmB promote ACLY palmitoylation, while the palmitoylation inhibitor 2BP inhibits ACLY palmitoylation, reducing ACLY enzyme activity, leading to decreased de novo lipid synthesis, reduced lipid deposition, and alleviated the symptoms of steatosis.

[0012] The inventors of this application evaluated the potential value of the palmitoylation inhibitor 2BP in the treatment of fatty liver disease through experimental studies, and provided the use of 2BP to inhibit ACLY palmitoylation and thus reduce lipid synthesis, providing new methods and new ideas for the clinical treatment of MASLD.

[0013] The present invention is achieved by the following technical solution: liver tissues of mice fed with a high-fat diet for 16 / 20 / 24 weeks were collected and the overall palmitoylation level of tissue proteins was detected by acyl-biotin exchange method. The results showed that as the number of weeks of high-fat diet feeding increased, the overall palmitoylation level of liver tissue proteins gradually increased (e.g. Figure 1 A); 1200 proteins were screened for palmitoylation modification by acyl-resin-assisted capture combined with mass spectrometry. Considering the important role of ACLY in lipid metabolism, we focused on ACLY for research (as shown in Figure 1 B); then, liver tissues of mice fed a high-fat diet were collected or human liver cancer cell HepG2 cells were treated with palmitic acid to confirm that ACLY could undergo palmitoylation modification (as shown in Figure 1 C). When HepG2 cells were treated with the palmitoylation agonist PalmB, the expression level of ACLY protein remained unchanged, but the palmitoylation level increased (as shown in Figure 2 As shown in A); when HepG2 cells were treated with the palmitoylation inhibitor 2BP, no significant change in ACLY protein expression was detected, and the palmitoylation level was significantly reduced (as shown in Figure 2 B). After treating cells with 2BP, the enzyme activity of ACLY was detected, and it was found that the enzyme activity of ACLY decreased after depalmitoylation (as shown in FIG. Figure 3Furthermore, the expression levels of proteins required for de novo lipid synthesis were detected by Western blotting. PA treatment increased the expression of phosphorylated ACLY, FASN, ACC and other proteins, and the addition of 2BP reversed the above effect, that is, the levels of phosphorylated ACLY, FASN, and ACC decreased (as shown in Figure 2A). Figure 3 BODIPY staining revealed that the number of intracellular lipid droplets in the 2BP-treated group decreased and lipid deposition was alleviated (as shown in Figure 2B). Figure 3 C).

[0014] The advantages of the present invention are: the present invention provides the use of 2BP for treating fatty liver disease associated with metabolic dysfunction; in particular, it is confirmed through animal and cell experiments that 2BP can inhibit the palmitoylation of ACLY, reduce de novo lipid synthesis, and thus alleviate lipid deposition. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 High-fat diet feeding leads to increased global palmitoylation levels of liver tissue proteins in mice

[0016] Among them, NCD: Normal Chow Diet; HFD: High Fat Diet; HAM: Hydroxylamine; Palm: Palmitoylation; A-RAC: Acyl-Resin Assisted Capture; ABE: Acyl-Biotin Exchange.

[0017] Compared with the normal diet group: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

[0018] Figure 2 2BP inhibits palmitoylation of ACLY

[0019] Among them, CON: Control, control group; PalmB: palmitoylation agonist; 2BP: palmitoylation inhibitor.

[0020] Compared with the control group: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

[0021] Figure 3 2BP reduces ACLY enzyme activity and lipid synthesis

[0022] Among them, P: Phosphorylation, phosphorylation; FASN: Fatty Acid Synthase, fatty acid synthase; ACC: Acetyl-CoA Carboxylas, acetyl-CoA carboxylase

[0023] Compared with the control group: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. It should also be understood that after reading the contents described herein, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] Example 1 High-fat diet feeding leads to increased overall palmitoylation levels of liver tissue proteins in mice

[0026] 1. Materials and Methods

[0027] 1. Animal Model and Treatment: Six-week-old C57 / BL6J male mice were fed a standard diet (NCD) or a high-fat diet (HFD, 60% kal, D12492, Research Diets Inc.) for 24 weeks to establish the MASLD mouse model and control mice. Mice were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd.

[0028] 2. Cell culture: Human hepatoma cell lines HepG2 were purchased from the cell bank of the Chinese Academy of Sciences. The cells were cultured using DMEM high glucose medium (Source Culture) + 10% fetal bovine serum (NATOCOR) + double antibody (Zhongqiao Xinzhou).

[0029] 3. Acyl-biotin exchange method to detect palmitoylation level:

[0030] (1) Free thiol blocking: After cell lysis, all unmodified free thiol groups were blocked with N-ethylmaleimide (NEM) to prevent nonspecific labeling.

[0031] (2) Palmitoyl-specific dissociation: Adding hydroxylamine selectively cleaves the thioester bond of palmitoylated proteins, exposing the newly formed sulfhydryl group.

[0032] (3) Nascent thiol biotin labeling: Use thiol-reactive biotin reagent to covalently label the thiol exposed by hydroxylamine to achieve biotinylation of palmitoylated proteins.

[0033] (4) Affinity enrichment and detection: Biotin-labeled proteins are pulled down by streptavidin magnetic beads, and then qualitative and quantitative analysis is performed by protein immunoblotting and polyacrylamide gel electrophoresis or mass spectrometry using antibodies against the target protein.

[0034] 4. Detection of palmitoylation levels using acyl-resin-assisted capture method:

[0035] (1) Free thiol blocking: After cell lysis, all unmodified free thiol groups are blocked with an alkylating agent to prevent nonspecific binding.

[0036] (2) Palmitoyl-specific dissociation: Hydroxylamine was added to selectively cleave the thioester bond, exposing the newly formed sulfhydryl group (no HAM was added to the control group).

[0037] (3) Thiol resin capture: The lysate is incubated with a thiol-reactive resin to covalently bind the exposed thiol groups.

[0038] (4) Elution and detection: Elute the bound protein with a reducing agent (such as sodium dodecyl sulfate); detect the target protein by Western blotting or globally analyze the palmitoylation group by mass spectrometry.

[0039] 5. Palmitoylation mass spectrometry detection:

[0040] (1) Liver tissue from NCD and HFD mice was trypsinized and peptides were eluted using a C18 spin column. After drying, the peptides were resuspended in 200 μL of loading buffer and mixed with 100 μL of high-capacity streptavidin beads.

[0041] (2) The eluted peptides were combined with 50 mM iodoacetamide to block reduced cysteine residues (potential palmitoylation sites). The peptides were then further desalted using C18 Stage Tips and analyzed by LC-MS / MS.

[0042] 2. Results

[0043] 1. As the number of weeks of high-fat diet feeding increased, the overall palmitoylation level of liver tissue proteins in mice gradually increased (e.g. Figure 1 (as shown in A).

[0044] 2. Using acyl-resin-assisted capture combined with mass spectrometry, we screened 1,200 proteins that could undergo palmitoylation. Considering the important role of ACLY in lipid metabolism, we focused on ACLY for research (e.g. Figure 1 B).

[0045] 3. Then, liver tissues of mice fed a high-fat diet were collected or human liver cancer cell HepG2 cells were treated with 200uMPA of palmitic acid to detect palmitoylation levels. It was confirmed that ACLY can undergo palmitoylation modification (such as Figure 1C).

[0046] Example 22BP inhibits palmitoylation of ACLY

[0047] 1. Materials and Methods

[0048] 1. Cell culture: HepG2 cells were cultured for 24 hours using the method of Example 1. The complete medium was replaced with a starvation medium. After 12-16 hours, different drugs were added. Cells were treated with 10 μM PalmB and 100 μM PalmB for 24 hours and then harvested.

[0049] 2. Western Blot: Tissue samples were lysed using RIPA buffer (containing 1% phosphatase inhibitor and 1% protease inhibitor). Protein samples were separated by electrophoresis on SDS-PAGE gels. Following standard blocking procedures, transfer to a membrane, and incubation with primary and secondary antibodies, the samples were developed using chemiluminescence. The grayscale values of the target proteins were analyzed using ImageJ software. The following antibodies were used: HRP-Streptavidin (Proteintech), ACLY (Proteintech), and β-Actin (Proteintech).

[0050] 3. IP-ABE method to detect ACLY palmitoylation:

[0051] (1) Cells were collected as described above, and ACLY antibody was added to the extracted protein lysate, and the mixture was placed in a Ferris wheel at 4°C for 12 hours.

[0052] (2) Wash the magnetic beads three times with PBS containing 0.1% Tween, and add the antigen-antibody complex to the washed magnetic beads.

[0053] (3) Blocking free sulfhydryl groups: Use an alkylating agent (such as N-ethylmaleimide, NEM) to block all unmodified free cysteine sulfhydryl groups in the sample to prevent subsequent nonspecific labeling.

[0054] (4) Specific cleavage of palmitoylated bonds and exposure of new sulfhydryl groups: Treat the sample with hydroxylamine. Hydroxylamine can specifically hydrolyze the thioester bond on palmitoylated cysteine, removing the palmitoyl group and exposing a new reactive sulfhydryl group.

[0055] (5) Labeling newly exposed sulfhydryl groups: Add a biotin-tagged reagent that specifically reacts with sulfhydryl groups. This reagent will covalently bind to the sulfhydryl groups exposed by the hydroxylamine treatment in the previous step. These biotin-labeled sites represent the original palmitoylation sites.

[0056] (6) Detection: Palmitoylated proteins and their modification levels can be qualitatively or quantitatively analyzed using streptavidin-mediated techniques, such as avidin pull-down enrichment and detection of biotin-labeled proteins.

[0057] 2. Results

[0058] 1. When HepG2 cells were treated with the palmitoylation agonist PalmB, no significant changes in ACLY protein expression were detected, but the palmitoylation modification level increased (e.g. Figure 2 A);

[0059] 2. When HepG2 cells were treated with the palmitoylation inhibitor 2BP, no significant changes in ACLY protein expression were detected, and the palmitoylation modification level was reduced (e.g. Figure 2 B).

[0060] Example 32BP reduces ACLY enzyme activity and lipid synthesis

[0061] 1. Materials and Methods

[0062] 1. Western blotting: As in Example 2, the required antibodies P-ACLY, FASN, ACC, and HMGCR were purchased from Proteintech.

[0063] 2. BODIPY staining: seed an appropriate amount of cells onto a confocal dish, starve them for 24 hours, collect the cells and fix them with 4% paraformaldehyde for 10 minutes, incubate them with BODIPY493 / 503 dye at 4°C in the dark for 30 minutes, stain the cell nuclei with DAPI for 2-5 minutes, and observe lipid droplets using a confocal microscope.

[0064] 3. ACLY enzyme activity detection:

[0065] (1) Collect cells: according to the number of cells or bacteria (10 4 The ratio of cells or bacteria to extract volume (mL) is 500-1000:1 (it is recommended that 5 million cells or bacteria be added to 1 mL of extract). Disrupt the cells or bacteria by ultrasonication in an ice bath (power 300 W, ultrasonication for 3 seconds, intervals of 7 seconds, total time 3 minutes). Centrifuge at 8000g for 10 minutes at 4°C. Collect the supernatant and place it on ice for testing.

[0066] (2) Preheat the spectrophotometer / enzyme reader for more than 30 minutes, adjust the wavelength to 340 nm, and zero with distilled water.

[0067] (3) Place reagent 1 in a 37°C water bath for 10 minutes.

[0068] (4) Operation table: Add the following reagents to a 96-well UV plate, mix thoroughly, and measure the absorbance value A1 at 340 nm after 10 seconds. Place the plate in a 37°C water bath or incubator for 2 minutes (the microplate reader has a temperature control function and can adjust the temperature to 37°C). Take it out, wipe it dry, and measure the absorbance value A2 at 130 seconds. Calculate ΔA: assay tube = A1 assay - A2 assay, ΔA blank tube = A1 blank - A2 blank, and ΔA = ΔA assay tube - ΔA blank tube (only 1-2 times are required for the blank tube).

[0069] Reagent Name (ul) Measurement tube (ul) Blank tube (ul) Reagent 1 152 152 Reagent 2 4 4 Reagent 3 20 20 Reagent 4 4 4 Reagent 5 10 10 sample 10 - distilled water - 10

[0070] (5) Calculate ACLY activity based on sample protein concentration: Unit definition: One unit of enzyme activity is defined as 1 nmol NADH consumed per mg protein per minute. ACLY activity (U / mg protein) = [ΔA × V total × 109 ÷ (ε × d)] ÷ (V sample × Cpr) ÷ T = 2679.5 × ΔA ÷ Cpr

[0071] 2. Results

[0072] 1. After treating cells with 2BP, the enzyme activity of ACLY was detected and it was found that the enzyme activity of ACLY decreased after depalmitoylation (e.g. Figure 3 (as shown in A).

[0073] 2. Western blotting was used to detect the expression levels of proteins required for de novo lipid synthesis. PA treatment increased the expression of phosphorylated ACLY, FASN, ACC and other proteins, and the addition of 2BP reversed the above effect, that is, the levels of phosphorylated ACLY, FASN, and ACC decreased (e.g. Figure 3 B).

[0074] 3. BODIPY staining and laser confocal microscopy revealed that the number of intracellular lipid droplets in the 2BP-treated group decreased and lipid deposition was alleviated (e.g. Figure 3 C).

[0075] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Application of palmitoylation inhibitors in the treatment of metabolic dysfunction-related fatty liver disease by inhibiting ACLY palmitoylation.

2. The use according to claim 1, characterized in that The palmitoylation inhibitor is 2BP.

3. The use according to claim 1, characterized in that The fatty liver disease is fatty liver disease associated with metabolic dysfunction.

4. The use according to claim 2, characterized in that The 2BP inhibits palmitoylation of ACLY.

5. The use according to claim 2, characterized in that The 2BP reduces ACLY enzyme activity, decreasing lipid synthesis.

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