CN114591418A

CN114591418A - Threonine 166 th phosphorylation modification of PPAR gamma protein and application thereof

Info

Publication number: CN114591418A
Application number: CN202011413315.0A
Authority: CN
Inventors: 沈萍萍; 杨南飞; 王于昕
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-06-07

Abstract

The invention belongs to the field of biological medicine, and relates to application of PPAR gamma 166 th site threonine phosphorylation modification as a disease marker in preclinical or clinical diagnosis and detection; the application in the staging, grading and prognosis evaluation of the disease course; the application in guiding the selection of clinical drugs; the application of the test paper or chip in the preparation of test kits, test paper or chips as research indexes; the application of the protein as a regulation target in the development of preparing medicaments for treating diseases; can be used as a regulation target point for treating metabolic diseases and immune system diseases. The invention is based on various biochemical and molecular biological means, identifies the 166 th threonine phosphorylation modification of PPAR gamma of mammals (including human beings), and confirms the functional position of the PPAR gamma in a metabolic system and an immune system. Meanwhile, the invention develops related detection reagents according to discovered biological functions, and screens and optimizes the micromolecule drug which can be used for regulating and controlling the phosphorylation modification by taking the phosphorylation modification as a target point.

Description

Threonine 166 th phosphorylation modification of PPAR gamma protein and application thereof

Technical Field

The invention belongs to the field of biological medicine, relates to application of PPAR gamma Thr166 phosphorylation modification as a drug marker for metabolic diseases, and relates to a treatment method for regulating metabolic disorders by interfering PPAR gamma Thr166 phosphorylation and small molecule compound information for interference.

Background

PPAR gamma is a drug target for clinical treatment of type II diabetes mellitus, and is also a key control factor for regulating and controlling various metabolic diseases, inflammations and tumors. The Pparg gene can form two variants by promoter selection: PPAR gamma₁And PPAR γ₂。 PPARγ₂

Than PPAR gamma

₁30 more amino acids are added at the N terminal, and the rest sequences are completely consistent. Generally, we refer collectively to PPAR γ, and the amino acid site information refers generally to PPAR γ₂The corresponding amino acid position in (a). (hereinafter, collectively referred to as PPAR. gamma. for convenience of description, the number of amino acid sites represents PPAR. gamma.)₂Amino acid site of (1)

The activation of PPAR gamma can promote the expression of insulin sensitivity gene and glycolipid metabolism related gene in cells and inhibit the expression of inflammatory related gene. The complete agonist Thiazolidinediones (TZDs) of PPAR gamma can remarkably enhance the insulin sensitivity of type II diabetes patients and reduce the blood sugar. At the same time, the agonism can reduce the infiltration of inflammatory cells in metabolic disorder organs and tissues and enhance the activity of immunoregulatory cells, thereby maintaining the metabolic and immune homeostasis of the body. However, TZDs, such as rosiglitazone, cause strong side effects, mainly including: retention of water and sodium, weight increase, cardiovascular and cerebrovascular problems, etc. The lack of study of the biological function of the drug target is reflected behind these phenomena. Therefore, searching for a control switch specifically activated by PPAR γ and designing, screening and developing novel small molecule modulators based on the control switch can lead to innovation of treatment of related diseases.

Phosphorylation is the most prominent post-translational modification of proteins. Proteins can be endowed with diverse biological functions by phosphorylation. Therefore, designing a targeting drug and regulating the post-translational modification, especially phosphorylation modification, of the protein is a brand-new drug development idea. The targeting process has the advantages that the protein target can be activated aiming at specific diseases, and the side effects caused by the traditional complete agonistic drugs can be avoided.

PPAR γ belongs to a member of the Nuclear Receptor (NRs) family, having four domains typical of NR: an N-terminal activation region, a DNA Binding Domain (DBD), a Hinge region (Hinge domain), and a Ligand Binding Domain (LBD). Previous studies on posttranslational modifications of PPAR γ have focused on LBD, and various posttranslational modifications have been discovered, including Ser273 phosphorylation, Lys268/293 acetylation, and Lys395 SUMO modification. However, the modified function of the DBD region and the application of the DBD region in disease diagnosis and drug development are still blank.

The pathogenesis of metabolic diseases is complex, and the combined action of multiple organs, multiple systems and multiple factors is involved. Clinically, the diagnosis of metabolic diseases mostly depends on serological indicators, but the serological indicators cannot accurately indicate the disease course state of the metabolic diseases. However, the prior art has not provided relevant molecular diagnostic markers, and appropriate targets and intervention strategies for guiding medication.

Disclosure of Invention

The first objective of the invention is to provide threonine phosphorylation at position 166 of PPAR gamma protein (corresponding to PPAR gamma)₁Middle site is threonine phosphorylation at position 136)

The second purpose of the invention is to provide a rabbit polyclonal antibody for detecting 166 th threonine phosphorylation of PPAR gamma protein and application thereof.

The third purpose of the invention is to provide a preparation method of the rabbit polyclonal antibody for detecting threonine phosphorylation at 166 th site of PPAR gamma protein.

The fourth purpose of the invention is to provide the application of phosphorylation of threonine 166 of PPAR gamma protein as a disease marker in preclinical or clinical diagnosis and detection.

The fifth purpose of the invention is to provide the application of the 166 th threonine phosphorylation of PPAR gamma protein as a target spot in drug screening, design and optimization.

The sixth purpose of the invention is to provide the phosphorylation of threonine 166 of PPAR gamma protein as an intervention target, and realize the application in the treatment of metabolic diseases by regulating the target.

The seventh purpose of the invention is to provide the application of the 166 th threonine phosphorylation of the PPAR gamma protein as a research index in the preparation of a detection kit, test paper or chip.

The 166 th threonine of the PPAR gamma protein is phosphorylated, the upstream kinase is PKC (including PKC alpha/beta/gamma), namely PKC can be bound on PPAR gamma, and the 166 th threonine of PPAR gamma is phosphorylated (located in a DNA binding region). The activity evaluation criteria was the determination of the transcriptional activity of PPAR γ.

The PPAR gamma protein 166 th threonine phosphorylation antibody is a mouse monoclonal antibody or a rabbit polyclonal antibody; the preparation method of the rabbit polyclonal antibody comprises the following steps:

1) polypeptide synthesis

2) Animal immunization: 2 healthy rabbits are selected, and 50-70 mL/rabbit of serum is collected after 2 mg/rabbit and 5 times of immunization;

3) identification of immune serum: using 10 mug/mL antigen coating and ELISA method to identify the serum titer;

4) purification of the antibody: affinity purifying the antibody by using a medium coupled with a phosphorylated peptide segment;

5) the specificity of the polyclonal antibody is determined by Western blotting.

The application of the antibody for phosphorylation of threonine 166 of PPAR gamma protein in detecting the phosphorylation level of threonine 166 of PPAR gamma protein in a cell or organ tissue sample. The antibody can also be used for developing detection entities such as a kit, test paper or a chip for detecting the 166 th threonine phosphorylation of the PPAR gamma protein.

The 166 th threonine phosphorylation of the PPAR gamma protein is used as a disease marker and is applied to preclinical or clinical diagnosis and detection. Detecting a disease marker as a metabolic disease, comprising: obesity, diabetes, adipose tissue metabolism disorder, adipocyte differentiation disorder, atherosclerosis, metabolic inflammation, tumor, metabolic kidney disease, metabolic liver disease, etc.; for evaluating PKC activation, PPAR gamma phosphorylation level, PPAR gamma transcriptional activity and metabolic state; for staging, grading and prognostic assessment of the course of the disease; for guiding the selection of clinical drugs; used for guiding the development of drugs capable of inhibiting the phosphorylation of threonine 166 of PPAR gamma.

The PPAR gamma protein 166 th site threonine phosphorylation is used as a target spot, and the application in drug screening, design and optimization comprises screening and design of a PPAR gamma ligand small molecule, a short peptide or an enzyme capable of inhibiting the phosphorylation site. Meanwhile, aiming at the upstream kinase, small molecule activators designed to inhibit PKC activation are screened.

The 166 th threonine phosphorylation of the PPAR gamma protein is used as an intervention target, and the application in the treatment of metabolic diseases is realized by regulating and controlling the target. After PPAR gamma threonine 166 is phosphorylated, the expression level of brown adipocyte markers in adipocytes is reduced, and the levels of lipid catabolism, fatty acid oxidative metabolism and mitochondrial oxidative phosphorylation are all inhibited. The intervention means comprises:

1) the phosphorylation sites of PPAR gamma can be interfered by chemical small molecules CDDO and CDDO-Im, namely the two small molecule compounds can be used as ligands of PPAR gamma to be combined with PPAR gamma, and the molecular conformation of PPAR gamma is changed to block the combination of an upstream kinase PKC and PPAR gamma, so that the 166 th threonine phosphorylation of PPAR gamma protein is inhibited, the brown fat marker expression of fat cells is promoted, the lipid catabolism is promoted, the fatty acid oxidative metabolism is promoted, and the mitochondrial respiration activity and the oxidative phosphorylation capability are enhanced.

2) PKC kinase activity is inhibited by using a PKC inhibitor Ro 31-8220 or the like, so that threonine phosphorylation at 166 th position of PPAR gamma is inhibited. The purpose of regulating and controlling the lipid metabolism and the mitochondrial function of the cells is achieved.

3) The Pparg gene is subjected to site-directed mutagenesis in a cell by using a gene editing technique, or the PPAR γ mutant protein is overexpressed in a cell by using a gene overexpression technique. By these techniques, a protein having the 166 th threonine mutation of PPAR γ was produced in cells. When the 166 th threonine of the PPAR gamma is mutated into Alanine (Alanine, A mutation), the continuous dephosphorylation state of the site can be simulated, and at the moment, the cellular lipid catabolism is vigorous, and the mitochondrial respiration and oxidative phosphorylation capacities are strong; when the 166 th threonine of PPAR gamma is mutated into Aspartic acid (D mutation), the continuous phosphorylation state of the site can be simulated, and the cellular lipid catabolism is inactivated, the lipid accumulation is strong, the mitochondrial respiration capacity is weakened, and the oxidative phosphorylation level is reduced.

4) The 166 th threonine phosphorylation of the PPAR gamma protein is regulated by other small molecular compounds, short peptides, antibodies or enzymes.

Through any one of the four ways, the targeted intervention on the phosphorylation level of threonine 166 th site of PPAR gamma in cells can be realized, the lipid metabolism steady state of the cells can be recovered, the mitochondrial state can be regulated, and various metabolic diseases can be further treated, including: obesity, diabetes, adipose tissue metabolism disorder, adipocyte differentiation disorder, atherosclerosis, metabolic inflammation, tumor, metabolic kidney disease, metabolic liver disease, etc.

Drawings

FIG. 1 is a mass spectrometric identification of PPAR γ phosphorylation at threonine 166; co-immunoprecipitation demonstrated the existence of an interaction between the upstream kinase PKC and the PPAR γ protein.

FIG. 2 shows that the mouse antibody with PPAR gamma 166 th threonine phosphorylation can specifically detect PPAR gamma 166 th threonine phosphorylation level in cells.

FIG. 3 is a graph showing that CDDO inhibits threonine phosphorylation at position 166 of PPAR γ in cells; CDDO interferes with the phosphorylation of PPAR γ by PKC.

FIG. 4 shows that phosphorylation of PPAR γ threonine at position 166 can be used as a diagnostic marker for metabolic diseases in an obesity model fed with high fat; mouse antibodies phosphorylated at threonine 166 can be used to detect the level of phosphorylation in tissues.

FIG. 5 shows that PPAR γ 166 th threonine phosphorylation is targeted and small molecule compounds capable of specifically inhibiting the phosphorylation are screened.

FIG. 6 shows that small molecular compounds CDDO and CDDO-Im interfere with phosphorylation of threonine 166 of PPAR γ in adipocytes, which can promote expression of brown fat-related genes and promote expression of genes related to lipid catabolism and fatty acid oxidative metabolism.

FIG. 7 shows that small molecular CDDO inhibits the phosphorylation of PPAR γ threonine 166 in animals, promotes the browning of adipose tissue, and increases the level of lipid catabolism; CDDO enhances mitochondrial number and mitochondrial respiratory activity within cells.

FIG. 8 shows that the 166 th threonine of PPAR γ is subjected to site-directed mutagenesis by gene editing technology, and the A mutation can enhance the transcriptional activity of PPAR γ, improve the lipid catabolic capacity of cells and enhance the mitochondrial respiratory activity. The D mutation has the opposite process.

FIG. 9 is a graph showing that metabolic disorder status can be improved by treating mice with the PKC inhibitor Ro 31-8220(Ro) in the metabolic disorder model of ob/ob transgenic mice.

FIG. 10 is a graph showing that the expression of insulin-sensitive genes in adipose tissue can be enhanced by treating mice with the PKC inhibitor Ro 31-8220(Ro) in a metabolic disorder model of ob/ob transgenic mice.

FIG. 11 is a graph showing that the classical activation phenotype of macrophages (inflammatory activation type M1) can be suppressed and the surrogate activation phenotype (anti-inflammatory phenotype M2) can be enhanced by treating mice with the PKC inhibitor Ro 31-8220(Ro) in a metabolic disorder model of ob/ob transgenic mice.

FIG. 12 is a graph showing that the protein having the 166 th threonine mutation of PPAR γ is overexpressed in macrophages by an overexpression exogenous gene technique, and the A mutation enhances the polarization of macrophage M2 while the D mutation enhances the polarization of macrophage M1.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The following detailed description is made with reference to the accompanying drawings in which:

FIG. 1 shows that the phosphorylation modification analysis of the reaction product of PKC and PPAR γ in the in vitro kinase reaction system by using phosphorylation mass spectrometry technology identifies a unique phosphorylation modification site, i.e., threonine phosphorylation modification at position 166 (FIG. 1. A-B). The interaction relationship between PKC and PPAR γ was subsequently analyzed in 293T model cells using co-immunoprecipitation techniques, indicating that PKC is an upstream kinase of PPAR γ in cells (fig. 1. C).

The preparation method of the polyclonal antibody of the embodiment is carried out according to the following steps: synthesizing polypeptide of PPAR gamma phosphorylated by amino acid sequence T166; and secondly, coupling the polypeptide synthesized in the step one with KLH to obtain immunogen, then immunizing healthy rabbits with the immunogen and collecting antiserum to obtain an antibody specifically recognizing phosphorylation of threonine 166. FIG. 2 shows the detection of threonine phosphorylation at position 166 of PPAR γ in both intracellular (FIG. 2.A, C) and in vitro kinase assays (FIG. 2.B) using the prepared antibodies.

FIG. 3 shows overexpression of wild-type PPAR γ in 293T cells and treatment with the ligand agonists RSG and CDDO of PPAR γ; the phosphorylation status was detected by using antibodies against threonine phosphorylation at position 166 of PPAR γ, and the results showed that CDDO at 50-100 μ M could effectively inhibit threonine phosphorylation at position 166 of PPAR γ (fig. 3. a). Further analysis of the interaction between PKC and PPAR γ after RSG and CDDO treatment using co-immunoprecipitation showed that 100 μ M CDDO was effective in blocking the interaction between PKC and PPAR γ (fig. 3. B).

Fig. 4 shows that the serum metabolic indicators were measured by feeding C57BL/6J mice with 60% fat diet for 2 months and 4 months: adiponectin (adiponectin), Triglyceride (TG), fasting glucose (blood glucose), total cholesterol (total cholesterol); metabolic inflammation markers: TNF- α and IL-6 (FIG. 4. A-C); meanwhile, detecting the phosphorylation levels of threonine 166 th position of PPAR gamma in white fat and brown fat by using Western blotting and the prepared antibody; it was found that the systemic metabolic disorder was also increased with the increase of obesity, and at the same time, the phosphorylation level of threonine at position 166 of PPAR γ in adipose tissue was gradually increased (FIG. 4.D-E), which indicates that the phosphorylation at this position can be used as a diagnostic marker for metabolic diseases.

FIG. 5 shows the screening of small molecules that directly inhibit the phosphorylation of PPAR γ threonine 166. FIG. 6.A shows that wild-type PPAR γ is overexpressed in 293T cells and treated with kinase inhibitors, and as a result, it was found that the activity inhibitor Ro 31-8220(Ro) of PKC can inhibit threonine phosphorylation at position 166 of PPAR γ (FIG. 5. A). Further, screening a PPAR gamma ligand small molecule capable of inhibiting the phosphorylation of PPAR gamma threonine 166 by using an in vitro kinase experiment; the results indicate that CDDO is a potent small ligand molecule that can inhibit this phosphorylated PPAR γ (fig. 5. B). This section of data demonstrates that PPAR γ phosphorylation at threonine 166 can be targeted, small molecule compounds that specifically inhibit this phosphorylation can be screened, and related drug molecules for the treatment of metabolic diseases can be developed.

FIG. 6 shows that when CDDO or CDDO-Im (FIG. 6.A) which is a structural analogue thereof is used to treat adipocytes in vitro, interference of CDDO and CDDO-Im with phosphorylation of threonine at position 166 of PPAR γ of adipocytes can promote expression of brown fat-related genes and expression of genes related to lipid catabolism and fatty acid oxidative metabolism (FIG. 6. B-E). [0041] The data for both parts [0042] indicate inhibition by the upstream kinase PKC; or the chemical small molecule ligands CDDO and CDDO-Im bind to PPAR gamma, and prevent the binding of the upstream kinase PKC and PPAR gamma; both can inhibit PPAR gamma 166 th phosphorylation modification, further promote fat cell brown fat marker expression, promote lipid catabolism, promote fatty acid oxidative metabolism and enhance mitochondrial respiration activity and oxidative phosphorylation capability.

FIG. 7 is an intraperitoneal injection of 4mg/kg CDDO into male C57BL/6J mice of 6-8 weeks of age, together with a positive control drug of Rosiglitazone (RSG) at 10mg/kg, once a day for 2 weeks; the body weight changes of the mice during the administration were monitored simultaneously, and it can be seen that CDDO inhibits the body weight gain of the mice (fig. 7. a). After dosing was complete, mice were sorted for three major adipose tissues: brown Adipose Tissue (BAT), Epididymal Adipose Tissue (EAT), and Subcutaneous Adipose Tissue (SAT); the content of the adipose tissues of the type can be compared by manually weighing the adipose tissues and converting the weight of the adipose tissues into the percentage of the adipose tissues in the body weight; as shown in fig. 7.B, CDDO specifically reduced the tissue content of subcutaneous adipose tissue. In parallel, the level of threonine phosphorylation at position 166 of PPAR γ was detected in subcutaneous adipose tissue, and it can be seen that the treatment with CDDO administration can significantly reduce the level of threonine phosphorylation at position 166 of PPAR γ in subcutaneous adipose tissue (fig. 7. C). Further analyzing the adipose tissue morphology, the protein distribution of the beige adipose marker UCP1 and the expression condition of the beige adipose cell biomarker in the subcutaneous adipose tissue by using H & E pathological sections, immunohistochemistry and fluorescent quantitative PCR technology (figure 7. E); the results show that CDDO can significantly promote the formation of a multi-compartmentalized morphology of subcutaneous adipose tissue (fig. 7.D), increase the expression level of UCP1 protein (fig. 7.G), and enhance the expression of beige adipocyte marker genes (fig. 7. F). On the other hand, by establishing an in vitro adipocyte differentiation model and treating cells with CDDO, it was found that CDDO can also inhibit the phosphorylation of threonine 166 of PPAR γ in adipocytes cultured in vitro (fig. 7.H), and strongly enhance the mitochondrial activity of adipocytes (fig. 7. I). The data in the part prove that the browning of the adipose tissue can be promoted and the lipid catabolism level can be promoted by intervening the phosphorylation of threonine 166 of PPAR gamma; can enhance the number of mitochondria in cells and the respiratory activity of mitochondria.

Fig. 8 is a mouse embryonic stem cell edited by CRISPR/Cas9 gene editing technology to obtain a transgenic mouse model with PPAR γ threonine 166 site-directed mutation, namely a mouse strain with alanine mutation (TA mutation) and aspartate mutation (TD mutation). The luciferase reporter system was used to detect the transcriptional activity of mutated PPAR γ, and it was found that the a mutation significantly enhanced the transcriptional activity of PPAR γ, while the D mutation significantly suppressed the transcriptional activity (fig. 8. B). Fat cells induced and differentiated in vitro are used, fat drops and mitochondria of the fat cells are detected through BODIPY 493/503 (neutral lipid fluorescent probe) and Mitotracker Red (mitochondrial membrane potential probe), and it can be seen that TA mutation can obviously improve the mitochondrial membrane potential of the fat cells and enhance the activity of the mitochondria; in contrast, the TD mutation significantly inhibited adipocyte mitochondrial viability (fig. 8. a). Further analyzing a gene expression profile through an RNA-Seq technology, finding that the fat cells with TA mutation show phenotypes of high lipid decomposition and high fatty acid oxidative metabolism; whereas TD is exactly the opposite (fig. 8. C). Fluorescent quantitative PCR also confirmed this conclusion (fig. 8. D). Furthermore, the respiratory capacity of wild WT, TA mutant and TD mutant adipocytes mitochondria was analyzed and detected using a Seahorse XF24 excellular fluorze energy metabolism analyzer, and the results showed that TA can greatly improve the respiratory activity of adipocytes mitochondria, while TD completely inhibits the mitochondrial respiratory activity of adipocytes (fig. 8. E). This section of data shows that Pparg is site-directed mutated in cells by using gene editing techniques or that PPAR γ mutant proteins are overexpressed in cells using gene overexpression techniques. By these techniques, a protein mutated by PPAR γ threonine 166 is produced in cells. When the 166 th threonine of the PPAR gamma is mutated into Alanine (Alanine, A mutation), the continuous dephosphorylation state of the site can be simulated, and at the moment, the cellular lipid catabolism is vigorous, and the mitochondrial respiration and oxidative phosphorylation capacities are strong; when the 166 th threonine of PPAR gamma is mutated into Aspartic acid (D mutation), the continuous phosphorylation state of the site can be simulated, and the cellular lipid catabolism is inactivated, the lipid accumulation is strong, the mitochondrial respiration capacity is weakened, and the oxidative phosphorylation level is reduced. Through gene editing or overexpression means, the targeted intervention on the phosphorylation level of threonine 166 th site of PPAR gamma in cells can be realized, the lipid metabolism steady state of the cells can be recovered, the mitochondrial state can be regulated, and then various metabolic diseases can be treated, including: obesity, diabetes, adipose tissue metabolism disorder, adipocyte differentiation disorder, atherosclerosis, metabolic inflammation, tumor, metabolic kidney disease, metabolic liver disease, etc.

FIG. 9 shows the metabolic disorder model of ob/ob transgenic mice treated with 2mg/kg of the PKC inhibitor Ro 31-8220(Ro) for 30 days by measuring the metabolic markers in serum (FIG. 9. A-I); and glucose tolerance and insulin resistance of the mice were measured to evaluate the insulin sensitivity status of the mice (fig. 9. J). This section of data demonstrates that inhibition of PKC activity can reduce insulin resistance and ameliorate systemic metabolic disorders.

FIG. 10 shows the administration of 2mg/kg of PKC inhibitor Ro 31-8220(Ro) for 30 days in a metabolic disorder model of ob/ob transgenic mice, followed by sorting white adipose tissues of the mice, and measuring the expression of genes related to insulin sensitivity in adipose tissues (FIG. 10.A), and the content of adiponectin in serum (FIG. 10. B); the results show that PKC inhibition can enhance insulin sensitivity of adipose tissues, increase the level of adiponectin in peripheral blood and obviously improve the systemic metabolic imbalance.

FIG. 11 is a pathological section of mice treated with 2mg/kg of the PKC inhibitor Ro 31-8220(Ro) for 30 days in a metabolic disorder model in ob/ob transgenic mice for analysis of adipose tissue immune cell infiltration; concurrently, mouse macrophages were sorted and the macrophage activation phenotype was determined (FIG. 11. A-B); the results indicate that PKC inhibition can improve infiltration of inflammatory immune cells, particularly macrophages; simultaneously inhibits the macrophage classical activation phenotype (inflammatory activation type M1), and enhances the alternative activation phenotype (anti-inflammatory phenotype type M2).

FIG. 12 shows the overexpression of the protein mutated by threonine 166 of PPAR γ in macrophages by the overexpression exogenous gene technique, and the detection of macrophage activation phenotype by the quantitative PCR technique (FIG. 12. A); simultaneously detecting the expression condition of the surface marker polarized by the macrophage and the phagocytic activity of the macrophage by using flow cytometry (figure 12. B-E); the results show that the a mutation enhances macrophage M2 polarization, while the D mutation enhances macrophage M1 polarization.

The above examples only represent several embodiments of the present invention, but should not be construed as limiting the scope of the invention. The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features. Without departing from the concept of the invention, several variations and modifications can be made, which are within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. It will be apparent to those skilled in the art that modifications and substitutions to methods, steps or conditions of the invention can be made without departing from the spirit and substance of the invention.

Claims

The application of PPAR gamma protein 166 th threonine phosphorylation as a disease marker in preclinical or clinical diagnosis and detection; the application in staging, grading and prognosis evaluation of disease course; use for guiding the selection of a clinical drug.
2. The use according to claim 1, wherein the disease type comprises metabolic diseases, including: obesity, diabetes, adipose tissue metabolism disorder, adipocyte differentiation disorder, atherosclerosis, tumor, metabolic kidney disease, metabolic liver disease, metabolic muscle disease, etc. The disease type also comprises immune system diseases caused by abnormal phosphorylation of threonine 166 of PPAR gamma protein, and the diseases comprise: metabolic inflammation, acute and chronic inflammation, immune cell or system dysplasia, autoimmune disease, allergy, tumor immunity abnormality, and the like. The detecting the sample comprises: serum plasma, tissue organ sample extracts, cell and cell in vitro cultures sorted from tissues or organs, and tissue organ specimen sections.
The application of the 166 th threonine phosphorylation of the PPAR gamma protein as a research index in preparing a detection kit, test paper or a chip.
The application of PPAR gamma protein 166 th threonine phosphorylation as a regulation target point in the preparation of drugs for treating diseases.
The use of the phosphorylation of threonine 166 of PPAR γ protein as a regulatory target in the treatment of metabolic and immune system diseases (the disease types are as in claim 1).
6. The mutant protein of the 166 th threonine site mutation of PPAR gamma of any mammal, or a recombinant expression vector of a corresponding coding gene thereof, and the application of the mutant protein and the recombinant expression vector in screening or preparing drugs (containing antibodies, polypeptides, enzymes and gene editing tools) for regulating and controlling the 166 th threonine site phosphorylation of PPAR gamma protein, or preparing detection kits, test paper or chips.
7.A recombinant host cell containing the mutant protein with PPAR gamma 166 th threonine site mutation of claim 5 or the corresponding coding gene thereof, and the application of the recombinant host cell in screening or preparing drugs, antibodies, polypeptides, enzymes and gene editing tools for regulating and controlling PPAR gamma 166 th threonine site phosphorylation.
8. Use of a mutant protein comprising a mutation at the 166 th threonine site of a PPAR γ protein according to claims 5 and 6, a corresponding recombinant expression vector, and a recombinant host cell comprising a protein according to claim 6 for the treatment of a metabolic disease or an immune system disease (the type of disease is as defined in claim 1).
9. The amino acid sequences of three human PPAR gamma 166 th threonine site mutant proteins are shown in a sequence table SEQ ID No.2 (alanine mutation), SEQ ID No.4 (aspartic acid mutation) and SEQ ID No.6 (glutamic acid mutation).
10. The DNA sequences of three coding genes of the mutant protein with the 166 th threonine site mutation of the human PPAR gamma are shown in a sequence table SEQ ID No.1 (alanine mutation), SEQ ID No.3 (aspartic acid mutation) and SEQ ID No.5 (glutamic acid mutation).
11. The amino acid sequences of three mouse PPAR gamma 166 th threonine mutant proteins are shown in a sequence table SEQ ID No.8 (alanine mutation), SEQ ID No.10 (aspartic acid mutation) and SEQ ID No.12 (glutamic acid mutation).
12. The DNA sequences of the three mouse PPAR gamma 166 th threonine site mutant protein coding genes are shown in a sequence table SEQ ID No.7 (alanine mutation), SEQ ID No.9 (aspartic acid mutation) and SEQ ID No.11 (glutamic acid mutation).
13. A mutant protein comprising a mutation at the threonine 166 th site of PPAR γ of claims 13-16, a recombinant expression vector encoding the mutant protein; a recombinant host cell containing a mutant protein with PPAR gamma threonine 166 th site mutation and a recombinant expression vector for coding the mutant protein; any one of the methods is applied to screening or preparing drugs (containing antibodies, polypeptides, enzymes and gene editing tools) for regulating and controlling phosphorylation of 166 th threonine site of PPAR gamma protein, or preparing detection kits, test paper or chips; use for the treatment of metabolic or immune system disorders (type of disorders as claimed in claim 1).
14. The use according to claim 1, the detection means comprising: antibody-dependent immunodetection means such as ELISA, Western blotting, Immunohistochemistry (IHC) and Immunofluorescence (IF) were performed on clinical samples using an antibody against threonine phosphorylation 166 th in PPAR γ protein (including all kinds of animal-derived antibodies, human recombinant antibodies, nanobodies, and the like). In addition, a nucleic acid analysis method for determining the 166 th mutation state by sequencing the PPAR γ gene is also included in the category.
15. The use according to claim 1 of diagnostic and detection means for indirectly determining the threonine phosphorylation state at position 166 of the PPAR γ protein by assessing the activation state of the upstream kinase PKC, comprising: using antibodies that detect PKC activation status, using in vitro kinase systems, or using other enzymatic activity assays. The diagnostic and detection samples are as described in claim 1.
16. The use according to claims 2, 5-7, and 12, wherein the 166 th threonine phosphorylation of PPAR γ protein is used as a detection index for developing a reagent for detecting an animal or human sample, or a product for commercial use such as a kit, a test paper, and a chip; the method specifically comprises the following steps: antibodies, Western blotting kits, ELISA kits, tissue chips, gene chips and the like. A gene editing reagent or kit for editing the 166 th threonine site of PPAR γ protein in animal or human cells or tissues; the method specifically comprises the following steps: a zinc finger editing tool, a TALEN editing tool, a CRISPR/Cas9 gene editing tool and a single base editing tool of the same family aiming at the locus, and a plasmid vector, a virus infection tool and the like prepared by the single base editing tool. Meanwhile, the application of the kit in claim 4, wherein the kit is used for detecting the 166 th threonine site of the PPAR gamma protein and animal or human samples, and developing detection services and detection kits for site mutation sequencing detection.
17. Use according to claims 3, 5-7, 12, characterized in that the phosphorylation of threonine 166 of the PPAR γ protein is used as a direct or indirect target for the modulation, in the preparation of a medicament for interfering with this phosphorylation level, in a manner comprising: drug screening, drug design, drug molecular structure optimization, and the like. The types of drugs include: a ligand small molecule compound, a short peptide, an antibody or an enzyme of PPAR γ that modulates the phosphorylation site. In addition, PKC activators/inhibitors are included that indirectly modulate this phosphorylation.
18. The use according to claims 4, 5-7 and 12, characterized in that the phosphorylation of threonine 166 of the PPAR γ protein is regulated by any intervention strategy and is used for the treatment of diseases. The regulation and control mode is realized by any one of the following modes:

1) the 166 th phosphorylation of PPAR gamma is regulated and controlled by chemical micromolecules CDDO and CDDO-Im and derivatives thereof;

2) through using PKC inhibitor to inhibit PKC kinase activity, the 166 th threonine phosphorylation of PPAR gamma is indirectly regulated;

3) site-directed mutagenesis of the coding sequence corresponding to threonine 166 of the gene of Pparg was carried out in cells by using gene editing techniques, or overexpression of a PPAR γ 166-threonine mutated mutant protein in cells by using gene overexpression techniques. By these techniques, a protein having the 166 th threonine mutation of PPAR γ is produced in cells or tissues and organs.

4) The 166 th threonine phosphorylation of the PPAR gamma protein is regulated by other small molecular compounds, short peptides, antibodies or enzymes.