Polypeptide for inhibiting binding of HNF4alpha and TRB3 protein and application of polypeptide in metabolic syndrome
By screening out peptides containing key amino acid sites and inhibiting the binding of TRB3 to HNF4ɑ protein, the problem of abnormal liver glucose and lipid metabolism was solved and the progression of metabolic syndrome was significantly inhibited.
Patent Information
- Application Number
- CN202310893000.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-20
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art has failed to effectively inhibit the binding of TRB3 to HNF4ɑ protein, resulting in abnormal liver glycolipid metabolism and promoting the progress of metabolic syndrome.
By screening out peptides containing key amino acid sites R164 and Q175, it inhibits the binding of TRB3 to HNF4ɑ protein and promotes the stability and transcriptional activity of HNF4ɑ protein.
Significantly inhibits the binding of TRB3 to HNF4ɑ, improves the stability and transcriptional activity of HNF4ɑ protein, inhibits fat deposition in liver cells, and alleviates the development of metabolic syndrome.
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Figure CN119331103A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology and relates to a polypeptide that inhibits the binding of HNF4ɑ to TRB3 protein and its application in treating metabolic syndrome-related diseases including non-alcoholic fatty liver disease, hyperlipidemia and diabetes. Background Art
[0002] Metabolic syndrome refers to a pathological state characterized by metabolic disorders of proteins, fats, carbohydrates, and other substances in the human body. It encompasses a complex set of metabolic abnormalities, including obesity, dyslipidemia, hypertension, and insulin resistance. It is an independent risk factor for cardiovascular and neurological diseases. The liver is the primary organ for the metabolism of nutrients such as proteins, fats, and carbohydrates. Endoplasmic reticulum stress in the liver can lead to inflammation and lipid deposition. Excessive lipid deposition, in turn, promotes endoplasmic reticulum stress and triggers autophagy in hepatocytes, further exacerbating damage. The interaction between endoplasmic reticulum stress and cellular damage mechanisms can affect insulin signaling, contributing to the pathogenesis of metabolic syndrome.
[0003] TRB3 is an important downstream effector molecule of endoplasmic reticulum stress. As a carrier protein that can bind to target proteins and E3 ubiquitinases, it has been proven to be involved in the occurrence and development of liver fibrosis and liver tumors, and is an important target for the treatment of liver-related diseases.
[0004] HNF4ɑ plays a crucial role in maintaining hepatocyte function and liver homeostasis. As a major transcription factor in the liver, HNF4ɑ regulates the expression of genes involved in glucose and lipid metabolism. Studies have shown that HNF4ɑ knockout mice develop spontaneous hepatic steatosis and inflammation, and that point mutations or loss of function in HNF4ɑ can directly lead to the development of human MODY type 1 diabetes. Using protein-protein interaction analysis and other related techniques, we identified TRB3 as a key partner that directly binds to HNF4ɑ and promotes its ubiquitination and degradation. Protein-protein docking and other methods further identified key amino acid sites that influence TRB3-HNF4ɑ binding. Based on this, we screened for a functional peptide containing these key sites that inhibits TRB3-HNF4ɑ binding and HNF4ɑ degradation. This peptide could be used to treat metabolic syndrome caused by a high-fat, high-sugar diet.
[0005] So far, there are no reports on the use of blocking or inhibitors that affect the binding of TRB3 to HNF4ɑ protein. Summary of the Invention
[0006] The purpose of the present invention is to provide a polypeptide that blocks or inhibits the binding of HNF4ɑ to TRB3 protein based on the above research results, and to provide the use of the polypeptide in the treatment of metabolic syndrome, in particular in the treatment of metabolic syndrome caused by non-alcoholic steatohepatitis and a high-fat, high-sugar diet.
[0007] After extensive and in-depth research, the inventors discovered for the first time that TRB3 protein can directly bind to HNF4ɑ protein and promote its degradation, leading to abnormal liver glucose and lipid metabolism and the progression of metabolic syndrome. Further, through a series of protein-protein interaction techniques, the key amino acid sites that affect the interaction between TRB3 and HNF4ɑ protein were identified: R164 / Q175 of TRIB3 and K300 / E353 of HNF4ɑ. Mutating the relevant sites in both proteins significantly inhibited the binding of TRB3 to HNF4ɑ and promoted the stability and transcriptional activity of HNF4ɑ protein. Based on this, a functional peptide containing the key binding sites (R164 and Q175) was screened based on the TRIB3 sequence. This peptide significantly inhibited the binding of TRB3 to HNF4ɑ, significantly inhibited the degradation of HNF4ɑ protein both in vitro and in vivo, suppressed fat deposition in hepatocytes in vitro and in vivo, and inhibited the development of metabolic syndrome in mice induced by a high-fat, high-glucose model.
[0008] In a first aspect, the present invention provides a polypeptide for inhibiting the binding of HNF4ɑ to TRB3 protein, wherein the polypeptide comprises a transmembrane peptide, a connecting peptide and an effector peptide.
[0009] The amino acid sequence of the membrane-penetrating peptide is as follows: HLYVSPW (SEQ ID NO.1); the connecting peptide is a connecting dipeptide (GG) formed by two glycines, and the amino acid sequence of the effector polypeptide is EPEAAVLFRQMATALAHCHQ (SEQ ID NO.3), in which the arginine (R) at the ninth position and the glutamine (Q) at the twentieth position are key sites for mediating the binding of HNF4ɑ to TRB3 protein. The hydrogen bond formed between the two mediates the binding of HNF4ɑ to TRB3 protein.
[0010] The inventors found that the above polypeptide has a competitive effect with the intracellular TRB3 protein, which can inhibit the binding of the intracellular TRB3 protein to HNF4ɑ ( Figure 8 Specifically, it promotes the transcriptional activity of HNF4ɑ protein ( Figure 9 ); inhibit cellular lipotoxicity ( Figure 10 ); concentration-dependently alleviates the progression of non-alcoholic fatty liver disease (NAFLD) in mice.
[0011] The EPEAAVLFRQMATALAHCHQ in the effector peptide was mutated to alanine to form an effector peptide mutant peptide with the sequence EPEAAVLFAQMATALAHCHA, which did not compete with the intracellular TRB3 protein and did not affect the binding of TRB3 protein to HNF4ɑ. At the same time, the experimental results also showed that it had no effect on the above experiments.
[0012] The second aspect of the present invention provides the use of the above polypeptide in the preparation of a drug for treating metabolic syndrome.
[0013] Preferably, the drug for treating metabolic syndrome is a drug for treating metabolic syndrome caused by non-alcoholic steatohepatitis or a high-fat, high-sugar diet.
[0014] The third aspect of the present invention provides a pharmaceutical composition for treating metabolic syndrome, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the polypeptide described above.
[0015] The active component may be the above-mentioned polypeptide as the sole active component, or may be a combination of the polypeptide and other drugs that improve metabolism.
[0016] The beneficial protection and effects of the present invention are as follows:
[0017] The present invention utilizes a series of protein function research technologies such as protein interaction, docking, site mutation and peptide screening, and combines in vitro and in vivo experiments to determine that TRB3 is a key partner molecule that binds to HNF4ɑ and promotes its ubiquitination degradation. It also identifies the key amino acid sites that affect the binding of TRB3 to HNF4ɑ protein, and further screens out functional polypeptides containing the key sites that can inhibit TRB3-HNF4ɑ binding and maintain the stability of HNF4ɑ protein. This polypeptide can exert the biological effect of correcting liver glucose and lipid metabolism disorders and can be used in the clinical treatment of metabolic syndrome including fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Knockdown of TRB3 protein significantly inhibited the degradation of HNF4ɑ protein and promoted the expression of its downstream lipid metabolism-related genes. A is the RT-PCR result, and B is the immunoblotting result.
[0019] Figure 2 It is the result of the interaction between TRB3 and HNF4ɑ protein;
[0020] Figure 3 To screen potential amino acid sites that affect the binding between TRB3 and HNF4ɑ protein for molecular docking technology;
[0021] Figure 4 To mutate TRB3 related sites to inhibit its binding to HNF4ɑ protein;
[0022] Figure 5 To mutate the HNF4ɑ related sites to inhibit its binding to TRB3 protein;
[0023] Figure 6 To promote the protein activity of HNF4ɑ by mutating HNF4ɑ related sites;
[0024] Figure 7 is a schematic diagram of wild-type and mutant polypeptides comprising TRB3-R164 / Q175;
[0025] Figure 8 Compared with the TRB3 mutant peptide, the wild-type peptide can significantly inhibit the binding of TRB3 to HNF4ɑ;
[0026] Figure 9 Compared with the TRB3 mutant peptide, the wild-type peptide can significantly restore the decreased HNF4ɑ activity caused by TRIB3;
[0027] Figure 10 Compared with the TRB3 mutant peptide, the wild-type peptide significantly inhibited the cellular lipotoxicity induced by palmitic acid / oleic acid (PAOA);
[0028] Figure 11 Compared with the TRB3 mutant peptide, the wild-type peptide can significantly alleviate non-alcoholic fatty liver disease caused by a high-fat and high-sugar diet. DETAILED DESCRIPTION
[0029] The implementation of the present invention is described in detail below in conjunction with the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.
[0030] Example 1:
[0031] Wild-type HepG2 cells were transfected with small interfering TRIB3 and cultured at a rate of 5 × 10 5 The cells were seeded at a density of 100 μg / mL in a 6-cm culture dish. The cells were lysed with SDS lysis buffer every 12 hours to collect protein at the time points of 0 hour, 12 hours, and 24 hours, and then subjected to immunoblotting after high-temperature denaturation. At the same time, the cells were lysed with TRIZOL to collect RNA at the time points of 0 hour, 24 hours, and 48 hours, and HNF4ɑ downstream genes were detected by real-time PCR.
[0032] The results are as follows Figure 1 Results showed that knockdown of TRB3 protein significantly inhibited the degradation of HNF4ɑ protein ( Figure 1 B) and promote the expression of downstream lipid metabolism related genes ( Figure 1 A).
[0033] Example 2:
[0034] 2×10 6HEK293T cells were seeded in a 90mm culture dish and transfected with a V5-tagged HNF4ɑ overexpression plasmid and a FLAG-tagged TRIB3 overexpression plasmid 8 hours later. After 48 hours, the cells were lysed and the protein was collected. Anti-V5 or anti-FLAG IgG immunomagnetic beads were added, respectively, and the cells were shaken overnight at 4 degrees. The beads were repeatedly washed with IP buffer and denatured at high temperature before immunoblotting. The results showed that HNF4ɑ protein can bind to TRIB3 protein ( Figure 2 ).
[0035] Experimental Example 3:
[0036] Protein-protein docking experiments were performed using the known HNF4A-LBD protein domain (PDB: 4IQR) and the simulated TRIB3 three-dimensional structure (Alphafold). The results showed that two groups of amino acid hydrogen bonds may mediate the interaction between HNF4α and TRIB3, namely TRIB3-R164 and TRIB3-Q175; HNF4α-K300 and HNF4α-E353 ( Figure 3 ).
[0037] Experimental Example 4:
[0038] 2×10 6 HepG2 cells were seeded in 90mm culture dishes. After 8 hours, wild-type TRIB3 and mutant TRIB3-2A (R164→A164, Q175→A175) with FLAG tags were transfected respectively. After 48 hours, the cells were lysed to collect the protein, and anti-FLAG IgG immunomagnetic beads were added. The cells were shaken overnight at 4 degrees. The beads were repeatedly washed with IP buffer and denatured at high temperature before immunoblotting. The results showed that the TRIB3-2A mutation could significantly inhibit the binding of TRIB3 to HNF4ɑ ( Figure 4 ).
[0039] Example 5:
[0040] 2×10 6 HepG2 cells were seeded in 90mm culture dishes and co-transfected with Flag-TRIB3+pcDNA, Flag-TRIB3+V5-HNF4α, and Flag-TRIB3+V5-HNF4α-2A 8 hours later. After 48 hours, the cells were lysed and the protein was collected. Anti-V5 IgG immunomagnetic beads were added to all three groups, shaken overnight at 4 degrees, and the beads were repeatedly washed with IP buffer. After high-temperature denaturation, immunoblotting experiments were performed. The results showed that the V5-HNF4α-2A mutation significantly inhibited the binding of TRIB3 to HNF4α ( Figure 5 ).
[0041] Example 6:
[0042] 8×10 3 HEK293T cells were seeded in 96-well plates and transfected with pGL3-NINJ1-9p (HNF4α reporter gene), pRL-SV40 plasmid, TRIB3 plasmid, and HNF4α wild-type and HNF4α-2A mutant plasmids 24 hours later. Samples were collected using a Dual-Luciferase Reporter Kit and OD 200 readings were performed using a TECAN infinite F200 microplate reader. The firefly luciferase and Renilla luciferase values were read, respectively, and the firefly luciferase value was divided by the Renilla luciferase value. The quotients of each group were compared and analyzed. The results showed that the HNF4α-2A mutant promoted the transcriptional activity of HNF4α protein ( Figure 6 ).
[0043] Example 7:
[0044] The I-TASSER server was used to screen the α-helical structure in the TRIB3117-220 protein domain, where T3H2 (aa 156-175) contains the key amino acids Arg-164 and Gln-175 that affect the binding of TRIB3 to HNF4α. Figure 7 , a transmembrane peptide (HLYVSPW, SEQ ID NO. 1) and a connecting dipeptide (GG) were added to the N-terminus of the α-helix to synthesize P-T3H2, and a transmembrane peptide control (HLYVSPWGG, P-Ctrl, SEQ ID NO. 2), a wild-type polypeptide (HLYVSPWGGEPEAAVLFRQMATALAHCHQ, P-T3H2, SEQ ID NO. 4), and a mutant polypeptide P-T3H2-2A (HLYVSPWGGEPEAAVLFAQMATALAHCHA, SEQ ID NO. 5) in which R164 was mutated to A164 and Q175 was mutated to A175 were synthesized respectively.
[0045] Example 8:
[0046] 2×10 6HepG2 cells were seeded in 90mm culture dishes. After 24 hours, they were transfected with a Flag-tagged TRIB3 wild-type plasmid. After 8 hours, a control peptide (P-Ctrl, 10uM), a wild-type peptide (P-T3H2, 10uM), and a R164A / Q175A mutant peptide (P-T3H2-2A, 10uM) were added. After 24 hours, the cells were lysed and the protein was collected. Anti-Flag IgG immunomagnetic beads were added to all four groups, shaken at 4 degrees overnight, and the beads were repeatedly washed with IP buffer. After high-temperature denaturation, immunoblotting experiments were performed. The results showed that the P-T3H2 wild-type peptide inhibited the binding of TRIB3 to HNF4α, while the P-T3H2-2A mutant peptide had no significant effect ( Figure 8 ).
[0047] Example 9:
[0048] HepG2 cells were collected at 3×10 3 Cells were seeded at a density of 100 μg / mL in 96-well plates and transfected with pcDNA and Flag-TRIB3 plasmids, respectively. 24 hours later, pGL3-NINJ1-9p (HNF4α reporter gene) and pRL-SV40 plasmids were further transfected. 8 hours later, control peptide (Pep-Ctrl, 10 μM), wild-type peptide (P-T3H2, 10 μM), and R164A / Q175A mutant peptide (P-T3H2-2A, 10 μM) were added, respectively. 24 hours later, samples were collected using a Dual-Luciferase Reporter kit and OD562 readings were performed on a TECANinfinite F200 microplate reader. The firefly luciferase and Renilla luciferase related values were read, and the firefly luciferase value was divided by the Renilla luciferase value. The quotients of each group were compared and analyzed. The results showed that the wild-type P-T3H2 peptide promoted the transcriptional activity of HNF4α protein, while the P-T3H2-2A mutant peptide had no significant effect ( Figure 9 ).
[0049] Example 10:
[0050] HepG2 cells were grown at 4×10 3The cells were seeded at a density of 100 μg / mL in a six-well plate and transfected with TRIB3 plasmid. 8 hours later, pcDNA and Flag-TRIB3 plasmid were transfected. 8 hours later, a transmembrane peptide control (P-Ctrl, 10 μM), wild-type peptide (P-T3H2, 10 μM) and R164A / Q175A mutant peptide (P-T3H2-2A, 10 μM) were added, respectively. After stimulation with de-fatty acid fetal bovine serum and palmitic acid oleic acid (PAOA), the cells were fixed with paraformaldehyde 24 hours later, stained with Nile red dye, and mounted with DAPI mounting medium. The images were taken under a laser confocal microscope. The results showed that the wild-type P-T3H2 peptide significantly inhibited the lipotoxicity of the cells, while the mutant P-T3H2-2A peptide had no significant effect ( Figure 10 ).
[0051] Example 11:
[0052] Eight-week-old mice were fed a high-fat, high-sugar diet to induce non-alcoholic fatty liver disease (NAFLD). Six weeks later, the mice were randomly divided into four groups (n=8) and injected with a cell-penetrating peptide control (P-Ctrl, 5 mg / kg), wild-type peptide (P-T3H2, 1 mg / kg), wild-type peptide (P-T3H2, 5 mg / kg), and R164A / Q175A mutant peptide (P-T3H2-2A, 5 mg / kg) via the tail vein twice a week. Four weeks later, insulin resistance was assessed by glucose tolerance test (GTT). The mice were then killed and liver tissues were cryopreserved or prepared into paraffin sections and frozen sections for liver tissue morphology. Hematoxylin and eosin staining was used to observe liver tissue morphology, oil red O staining was used to assess lipid droplet deposition, liver tissue homogenates were used to assess total cholesterol (TC) and triglycerides (TG), and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured.
[0053] The results are as follows Figure 11 The results showed that the wild-type P-T3H2 peptide alleviated the progression of NAFLD in mice in a concentration-dependent manner, while the mutant P-T3H2-2A peptide had no significant effect.
[0054] From the above experimental results, it can be seen that through a series of protein function research technologies such as protein interaction, docking, site mutation and peptide screening, combined with in vivo and in vitro biochemical experiments, it is determined that TRIB3 protein is a key molecule that binds to HNF4α and inhibits its transcriptional activity, and the key amino acid sites that affect the binding of TRIB3 and HNF4α proteins and the function of TRIB3 protein are identified. Functional polypeptides that can inhibit TRIB3-HNF4α binding and protect HNF4α protein activity, including key sites, are further screened out. This polypeptide plays a biological role in inhibiting lipid droplet deposition in vivo and in vitro.
[0055] It lays the foundation for the development of small molecule drugs for the treatment of metabolic syndrome and is widely used in the adjuvant treatment of patients with metabolic syndrome.
[0056] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.
Claims
1. A polypeptide for inhibiting the binding of HNF4ɑ to TRB3 protein, characterized in that: The polypeptide comprises a membrane-penetrating peptide, a connecting peptide and an effector polypeptide, and the amino acid sequence of the effector polypeptide is shown in SEQ ID NO.
3.
2. The polypeptide according to claim 1, characterized in that: in, The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.
1.
3. The polypeptide according to claim 1, characterized in that: in, The connecting peptide is a connecting dipeptide formed by two glycines.
4. Use of the polypeptide according to claim 1 in the preparation of a drug for treating metabolic syndrome.
5. The use according to claim 4, characterized in that: in, The drug for treating metabolic syndrome is a drug for treating metabolic syndrome caused by non-alcoholic fatty hepatitis or a high-fat and high-sugar diet.
6. A pharmaceutical composition for treating metabolic syndrome, characterized in that: The invention comprises an active component and a pharmaceutically acceptable carrier, wherein the active component comprises the polypeptide according to claim 1.
7. The pharmaceutical composition for treating metabolic syndrome according to claim 6, characterized in that: The active component comprises the polypeptide according to claim 1 as the only active component.
8. The pharmaceutical composition for treating metabolic syndrome according to claim 6, characterized in that: The active component is a combination of the polypeptide according to claim 1 and other drugs that improve metabolism.