WO2016011906A1 - Use of ubiquitination pathway-related factors in regulating function of t helper cell - Google Patents

Abstract

Disclosed is a use of ubiquitination pathway-related factors in regulating the function of T helper cell. In particular, disclosed is a use of an ubiquitination pathway-related factor, an agonist or an antagonist thereof in the preparation of a preparation or a kit for regulating RORγt and T helper cells, wherein the ubiquitination pathway-related factor is selected from proteins such as USP4, USP17, and so on, encoded by a proto-oncogene of the deubiquitination protein family.

Description

Application of ubiquitination pathway related factors in the regulation of helper T cell function Technical field

The invention relates to the field of molecular biology and biomedicine. More specifically, the invention relates to the use of ubiquitination pathway-related factors in the regulation of helper T cell function.

Background technique

Helper Th17 cells are newly discovered CD4-positive effector T cell subsets that promote inflammatory responses in the immune system. They participate in T cell immune responses by producing the cytokine interleukin-17 (IL-17), coordinate the body's defense against specific pathogens, and regulate tissue inflammatory responses, playing an important role in infectious diseases, autoimmunity and transplant rejection. effect. The orphan nuclear receptor RORgammat (RORγt) is important for the differentiation and function of helper Th17, and is a specific transcriptional regulator of cell differentiation. It can also regulate Th17 inflammatory response by inducing IL-17 secretion.

Current studies indicate that the tumor growth factors TGF-β and interleukin-6 (IL-6) are essential for the differentiation of Th17, and interleukin 1beta (IL-1beta) and interleukin-23 (IL-23) are also required. Over-activation of pro-inflammatory Th17 cells is a key factor in the development of many inflammatory and autoimmune diseases. At present, there are many reports on the regulation of Th17 cells. The tumor necrosis factor receptor-associated factor 3 (TRAF3) family can negatively regulate IL-17 production by targeting the IL-17 receptor, and TRAF6 inhibits the differentiation of Th17.

Those skilled in the art have been working to develop techniques for modulating the activity of the immune system for the treatment of diseases caused by disorders of the immune response.

Summary of the invention

It is an object of the present invention to provide an application of a ubiquitination pathway-associated factor in the regulation of helper T cell function.

In a first aspect of the invention, there is provided the use of a deubiquitinating enzyme or a deubiquitinating enzyme agonist for the preparation of a formulation or kit for:

(1) increasing the stability of RORγt; and/or

(2) up-regulating helper T cell activity or promoting differentiation of helper T cells; and/or

(3) modulating the expression or activity of a helper T cell cytokine; and/or

(4) Treating and/or preventing diseases associated with hypoxia of helper T cells.

In another preferred embodiment, the helper T cell activity is too low to mean that the helper T cell pro-inflammatory function is too low.

In another preferred embodiment, the disease associated with hypoxia of helper T cells is selected from the group consisting of tumors and infectious diseases.

In another preferred embodiment, the tumor is selected from the group consisting of prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, stomach cancer, bladder cancer, skin cancer, Rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental villus cancer, glioma, lymphoma, leukemia, rectal adenocarcinoma or melanoma.

In another preferred embodiment, the infectious disease is selected from the group consisting of: plague, cholera, infectious atypical pneumonia, AIDS, viral hepatitis, polio, human infection with highly pathogenic avian influenza, measles, epidemic hemorrhagic fever , rabies, epidemic encephalitis, hand, foot and mouth disease, dengue fever, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid and paratyphoid fever, epidemic cerebrospinal meningitis, whooping cough, diphtheria, neonatal tetanus, Scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, influenza, epidemiology Mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, epidemic and endemic typhus, kala-azar, echinococcosis, filariasis, in addition to cholera, bacterial and amoebic dysentery, typhoid fever and paratyphoid fever Infectious diarrheal disease, or fungal infection.

In another preferred embodiment, the deubiquitinating enzyme is selected from one or more of the group consisting of: Ubiquitin-specific proteases (USPs) and the OUT family.

In another preferred embodiment, the deubiquitinating enzyme is selected from one or more of the group consisting of USP2, USP3, USP4, USP5, USP7, USP10, USP12, USP14, USP17, USP18, USP21, USP22, USP30, USP39, USP44, YOD1, CYLD and A20; preferably, the deubiquitinating enzyme is USP2, USP4, USP17 or a combination thereof.

In another preferred embodiment, the helper T cell is selected from one or more of the group consisting of Th1, Th2, Th3, Th9, Th17, and Tfh.

In another preferred embodiment, the helper T cell cytokine is selected from one or more of the group consisting of FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2.

In another preferred embodiment, the "modulating the expression or activity of a helper T cell cytokine" means:

Downregulating expression or activity of one or more helper T cell cytokines selected from the group consisting of IL-17A, IL-17F, IL-21, IL-23R and/or IL-2; and/or

Upregulate the expression or activity of FOXP3.

In another preferred embodiment, the deubiquitinating enzyme agonist is selected from one or more of the group consisting of a deubiquitinating enzyme nucleation promoter and/or an inflammatory cytokine, preferably, said The inflammatory cytokine is TGF-β.

In another preferred embodiment, the deubiquitinating enzyme nucleating promoter refers to a substance that promotes deubiquitinating enzyme entry into the nucleus or a substance that increases ubiquitinating enzyme content in the nucleus (such as IL6).

In another preferred embodiment, said increasing the stability of RORyt is achieved by deubiquitinating RORyt.

In another preferred embodiment, the deubiquitination is selected from the group consisting of: deubiquitination of the RORyt K48 site.

In another preferred embodiment, the deubiquitinating enzyme is derived from a mammal (selected from: human).

In another preferred embodiment, the amino acid sequence of the USP4 is as shown in SEQ ID NO.: 2;

The amino acid sequence of USP17 is set forth in SEQ ID NO.: 4; and/or

The amino acid sequence of the USP2 is shown in SEQ ID NO.: 6.

In another preferred embodiment, the deubiquitinating enzyme is selected from the group consisting of a wild-type or mutant deubiquitinating enzyme, and is also selected from the group consisting of deubiquitin having the same function as the wild-type deubiquitinating enzyme. An active fragment of an enzyme or a derivative of a deubiquitinating enzyme.

In another preferred embodiment, the derivative of the deubiquitinating enzyme is selected from the group consisting of: a modified deubiquitinating enzyme molecule, an amino acid sequence homologous to a natural deubiquitinating enzyme, and having natural deubiquitinating enzyme activity Protein molecule, dimer or multimer of deubiquitinating enzyme, fusion protein containing deubiquitinating enzyme amino acid sequence.

In another preferred embodiment, the modified deubiquitinating enzyme is a PEGylated deubiquitinating enzyme.

In another preferred embodiment, the "protein molecule having an amino acid sequence homologous to the natural deubiquitinating enzyme and having natural deubiquitinating enzyme activity" means that the amino acid sequence thereof is compared with the wild type amino acid sequence (eg, for USP4) In contrast, compared to SEQ ID NO.: 2, having > 85% homology, preferably > 90% homology, more preferably > 95% homology, optimally > 98 % homology; and a protein molecule having natural deubiquitinating enzyme activity.

In another preferred embodiment, the disease associated with helper T cell activity refers to a disease or condition associated with excessive or low helper T cell activity.

In another preferred embodiment, the disease associated with helper T cell activity is selected from the group consisting of a tumor, an inflammatory response, or an autoimmune disease.

In another preferred embodiment, the preparation is selected from the group consisting of a pharmaceutical composition, a nutraceutical composition, a food composition, a vaccine composition, or an experimental reagent.

In a second aspect of the invention, there is provided the use of a deubiquitinating enzyme antagonist for the preparation of a formulation or kit for:

(1) reducing the stability of RORγt; and/or

(2) down-regulating helper T cell activity or inhibiting differentiation of helper T cells; and/or

(3) modulating the expression or activity of a helper T cell cytokine; and/or

(4) treating and/or preventing a disease associated with an excessively high level of helper T cell activity; and/or

(5) up-regulating the expression of FOXP3, enhancing the immunosuppressive function of Treg; and/or

(6) Treating and/or preventing diseases associated with regulatory T cell activity.

In another preferred embodiment, the "modulating the expression or activity of a helper T cell cytokine" means:

Upregulating expression or activity of one or more helper T cell cytokines selected from the group consisting of IL-17A, IL-17F, IL-21, IL-23R and/or IL-2; and/or

Down-regulate the expression or activity of FOXP3.

In another preferred embodiment, the disease associated with hyperactivity of helper T cells is selected from the group consisting of an inflammatory response and an autoimmune disease.

In another preferred embodiment, the inflammatory response is selected from the group consisting of: allergic inflammation, folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, asthma, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, Inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, vasculitis or interstitial cystitis.

In another preferred embodiment, the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, rheumatoid arthritis, rheumatic heart disease, rejection in organ transplantation, systemic lupus erythematosus, gram Autoimmune diseases such as Ron's disease, ulcerative colitis, ankylosing spondylitis, autoimmune encephalomyelitis, psoriasis, and the like, ulcerative colitis and autoimmune encephalomyelitis.

In another preferred embodiment, the deubiquitinating enzyme antagonist is selected from the group consisting of an antibody, a sh-RNA, a miRNA, an antisense oligonucleotide, a chemical inhibitor, or a combination thereof.

In another preferred embodiment, the antagonist is selected from the group consisting of: an anti-USP4 antibody, an anti-USP17 antibody, shUSP4 (short hairpin RNA), shUSP17 (short hairpin RNA), an antisense oligonucleotide against USP4 or USP17, Chemical inhibitor of USP4 or USP17.

In another preferred embodiment, the nucleotide sequence of shUSP17 is set forth in SEQ ID NO.: 16.

In another preferred embodiment, the chemical inhibitor is: 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-di Baseline phenylacetic acid (Vialinin A).

In another preferred embodiment, the regulatory T cell is selected from the group consisting of: a naturally-regulating T cell (nTreg) and an induced adaptive regulatory T cell (iTreg).

In another preferred embodiment, the disease associated with regulatory T cell activity is selected from the group consisting of cancer, viral infection, autoimmune disease, preferably autoimmune disease.

In a third aspect of the invention, there is provided a composition for increasing the stability of RORyt selected from the group consisting of: a deubiquitinating enzyme or a derivative thereof, and/or a deubiquitinating enzyme agonist.

In another preferred embodiment, the deubiquitinating enzyme agonist is as described above.

In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of a solid formulation, a liquid formulation, preferably in the form of a dry powder or solution.

In another preferred embodiment, the composition is a pharmaceutical composition, a nutraceutical composition or a vaccine composition.

In a fourth aspect of the invention, there is provided an isolated complex which is a complex formed by the combination of a deubiquitinating enzyme and RORyt.

In another preferred embodiment, the complex is a binary complex.

In another preferred embodiment, the complex has a molecular weight of ≥ 80 KD; preferably the molecule of the complex The amount is ≥100KD.

In another preferred embodiment, the complex has a molecular weight of from 100 KD to 500 KD; preferably from 100 KD to 200 KD.

According to a fifth aspect of the invention, there is provided the use of the complex of the fourth aspect of the invention for screening a drug or a compound which promotes or inhibits deubiquitinating enzyme and RORγt to form said complex .

In another preferred embodiment, when the complex is used to screen for a drug, the application includes the steps of:

(a) in the test group, in the presence of the test substance, culturing the helper T cells, and setting a control group without the substance to be tested;

(b) Detecting the content H1 of the complex in the test group and comparing it with the complex content H0 in the control group, wherein when H1 is significantly higher than H0, it means that the test substance is a promoter of deubiquitinating enzyme. When H1 is significantly lower than H0, it means that the test substance is an antagonist of deubiquitinating enzyme.

In another preferred embodiment, when the complex is used to screen for a drug, the application includes the steps of:

(a) in the test group, co-incubating the test substance with (a) the complex and/or (b) deubiquitinating enzyme and RORγt; and setting a control group without the substance to be tested;

(b) Detecting the content H1 of the complex in the test group and comparing it with the complex content H0 in the control group, wherein when H1 is significantly higher than H0, it means that the test substance is a promoter of deubiquitinating enzyme. When H1 is significantly lower than H0, it means that the test substance is an antagonist of deubiquitinating enzyme.

In a sixth aspect of the invention, there is provided a use of a deubiquitinating enzyme antagonist for the manufacture of a medicament for the treatment of systemic lupus erythematosus or rheumatic heart disease.

In another preferred embodiment, the deubiquitinating enzyme antagonist is selected from the group consisting of an antibody, a sh-RNA, a miRNA, an antisense oligonucleotide, a chemical inhibitor, or a combination thereof.

In another preferred embodiment, the antagonist is selected from the group consisting of: an anti-USP4 antibody, an anti-USP17 antibody, shUSP4 (short hairpin RNA), shUSP17 (short hairpin RNA), an antisense oligonucleotide against USP4 or USP17, Chemical inhibitor of USP4 or USP17.

In another preferred embodiment, the nucleotide sequence of shUSP17 is set forth in SEQ ID NO.: 16.

In another preferred embodiment, the chemical inhibitor is: 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-di Baseline phenylacetic acid (Vialinin A).

In a seventh aspect of the invention, there is provided a use of a composition comprising one or both components selected from the group consisting of IL-6 and TGFβ for the preparation of a reagent for promoting deubiquitinating enzyme entry into the nucleus.

In another preferred embodiment, the deubiquitinating enzyme is selected from one or more of the group consisting of: Ubiquitin-specific proteases (USPs) and the OUT family.

In another preferred embodiment, the deubiquitinating enzyme is selected from one or more of the group consisting of USP2, USP3, USP4, USP5, USP7, USP10, USP12, USP14, USP17, USP18, USP21, USP22, USP30, USP39, USP44, YOD1, CYLD and A20; preferably, the deubiquitinating enzyme is USP2, USP4, USP17 or a combination thereof.

In another preferred embodiment, the agent that promotes deubiquitinating enzyme entry into the nucleus includes an agent that promotes deubiquitinating enzyme entry into the nucleus and/or an agent that increases ubiquitination enzyme content in the nucleus.

In an eighth aspect of the invention, there is provided 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-diphenyl phenylacetic acid, Use of a derivative thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of an autoimmune disease, tumor, or inflammatory response.

In another preferred embodiment, the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, rheumatoid arthritis, rheumatic heart disease, rejection in organ transplantation, systemic lupus erythematosus Autoimmune diseases such as Crohn's disease, ulcerative colitis, ankylosing spondylitis, autoimmune encephalomyelitis, psoriasis, preferably ulcerative colitis and autoimmune encephalomyelitis.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows that USP4 is highly expressed in Th17 and affects the stability of its important transcription factor RORγt.

Figure 1A, Figure 1B. Flow cytometry (FACS) sorted CD45RA+CD4+naive T cells from human peripheral blood cells (PBMC), and different helper T cells were obtained under different helper T cell induction conditions. Subpopulations and inducible regulatory T cells (iTreg), extracting RNA for specific primer real-time fluorescent quantitative PCR (q-RTPCR) or specific antibody Western blotting (WB), the results show that USP4 is highly expressed in Th17 . In the figure, shCK is a control in which the expression of the gene has no effect, and shUSP4-1 and shUSP4-2 are the experimental groups in which the USP4 gene is specifically knocked down.

Fig. 1C, Fig. 1D. Naive T cells were induced for 7 days under Th17 induction conditions, and incubated with USP4 gene knockdown virus (puromycin resistance) overnight. After transfusion, 72 days after infection, puromycin was added for one week, and cells were collected for q. -RTPCR and immunoblotting with the corresponding antibodies showed that knockdown of USP4 can affect the stability of RORγt protein level, but has no effect on its mRNA level, indicating that the regulation of RORγt by USP4 may be the level of post-translational modification.

Figure 2 shows that USP4 affects the stability of RORγt by binding and deubiquitination, wherein

Figure A. Naive T cells were induced for 7 days under Th17 induction, then cells were harvested and lysed by RIPA buffer, then co-immunoprecipitated with USP4 antibody, and then immunoblotted with the corresponding antibodies. The results showed that USP4 and RORγt were in Th17. Direct interaction; IP-USP4 indicates that USP4 protein is enriched in cell lysate with USP4 specific recognition antibody, and INPUT indicates supernatant of untreated cell lysate, the same below;

Figure B. Co-transformation of HA-USP4 or its enzyme-deficient mutant Ser311A mutant with FLAG-RORγt into HEK 293T cells, and treatment of cells with inhibitory protein synthesis reagent (CHX (Cycloheximide from microbial) cycloheximide) 36 h later At 4,8,12h, the cells were collected and lysed, and then HA and FLAG and other corresponding antibodies were used for immunoblot analysis. The results showed that wild type USP4 can prolong the half-life of RORγt protein, but the activity of the enzyme mutant is weak. HA-USP4 represents the HA-tagged USP4 protein expressed by linking the HA tag and the USP4 gene, and FLAG-RORγt represents the FLAG-tagged RORγt protein expressed by linking the FLAG tag and the RORγt gene together, and WT indicates wild type. , CA indicates that the cysteine at position 311 of the amino acid sequence of USP4 is mutated to alanine, and the protein conformation and hydrophobicity are changed, and the enzyme activity is decreased, the same below;

Figure C. HA-USP4 with FLAG-RORγt and His-ubiquitin or its mutant (Lys48only, all lysine on ubiquitin is mutated to arginine, except for the 48th retention, Lys63only All lysines on ubiquitin were mutated to arginine, except for the 63rd retention), which were co-transformed into HEK 293T cells. Cells were harvested 48h and denatured with 8M urea and then bound with nickel beads (beads). His (ie His-pull down assay), followed by immunoblotting analysis with FLAG and other corresponding antibodies, showed that the deubiquitination of RORγt by USP4 only targets the 48th lysine-dependent modification on ubiquitin, and this A specific position of ubiquitination has been shown to be involved in the degradation of proteins; His-ubi represents a His-tagged ubiquitin protein expressed by linking six His-tags to a ubiquitin gene;

Figure D. FLAG-RORγt-Jurkat stable strain was stimulated with USP4 inhibitor (20 nM) for 4 hours, and without stimulation and FLAG-Jurkat control, cells were harvested and lysed and then co-immunoprecipitated with FLAG, and then used. Immunoblot analysis of endogenous ubiquitin antibodies and other corresponding antibodies showed that inhibition of endogenous USP4 enzyme activity by the chemical reagent vialinin A affected the level of ubiquitination of RORγt.

Figure 3 shows that USP4 promotes RORγt-induced transcription and production of interleukin-17, wherein

Figure A. IL-17A luciferase promoter reporter gene (-600-0bp), Renilla (control) and different gradients of HA-USP4 and FLAG-RORγt co-transformed into HEK 293T cells, 48h collection of cells with fluorescein After the enzyme reporter lysate was lysed, the supernatant was added to the reaction solution and the blocking solution, and the fluorescence value was measured. Then, HA and FLAG and other corresponding antibodies were used for immunoblot analysis, and the results showed that USP4 mediated the transcription of IL-17A by RORγt. Promotion is USP4 gradient dependent;

Figure B, C. HEK 293T cells were transfected into USP4 gene knockdown plasmid (puromycin resistance). After transfection, 72 hours after transfection, puromycin was added for one week. Cells were passaged and transferred to IL-17A luciferase. The promoter reporter gene (-600-0bp), renilla (control) and FLAG-RORγt were collected. After 48 hours, the cells were collected for fluorescence measurement and immunoblotting with the corresponding antibody. The results showed that the endogenous USP4 was knocked down, and RORγt was mediated. The transcription level of IL-17A is inhibited;

Figure D, E. IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) were transferred into FLAG-RORγt-Jurkat or FLAG-Jurkat cells, and PMA and inomycin (muscle) were added after 44h. (P/I, simulated TCR signal), after 4 hours, the cells were collected and lysed with luciferase reporter lysate, and the supernatant was added to the reaction solution and the blocking solution to measure the fluorescence value, and then USP4 and FLAG and other corresponding The antibody was used for immunoblot analysis. The results showed that the addition of USP4 inhibitor in the T cell stably inhibited RORγt-mediated transcription of IL-17A promoted by the TCR signaling pathway.

Figure 4 shows that IL-6 promotes RORγt-induced interleukin-17 transcription and production by altering the nuclear localization of USP4,

Figure A. IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) was transferred into FLAG-RORγt-Jurkat or FLAG-Jurkat cells, and fresh juice was added after 36h (P/ I) overnight, after adding TGFβ (1 ng/ml) and IL-6 (20 ng/ml) for 8 hours, the cells were collected and lysed with luciferase reporter lysate, and the supernatant was added to the reaction solution and the blocking solution to measure the fluorescence value. Then USP4 and FLAG and other corresponding antibodies were used for immunoblot analysis. The results showed that the promotion of RORγt-mediated IL-17A transcription by TGFβ and IL-6 can be inhibited by USP4 (enzyme activity inhibitor (USP4i)). Offset for Vialinin A);

Figure B, C. FLAG-RORγt-Jurkat or FLAG-Jurkat cells were incubated with USP4 gene knockdown virus (puromycin resistance) overnight. After transfusion, 72 days after infection, add puromycin for one week and then transfer to IL-17A. The luciferase promoter reporter gene (-600-0bp), renilla (control), 36h later, fresh liquid was added (P/I) overnight, TGFβ (1ng/ml) and IL-6 (20ng/ml) were added. After 8 hours of stimulation, the cells were collected and lysed with luciferase reporter lysate. The supernatant was added to the reaction solution and the blocking solution, and the fluorescence was measured. Then, USP4 and FLAG and other corresponding antibodies were used for immunoblot analysis. The results showed that TGFβ and IL- 6 The promotion of RORγt-mediated IL-17A transcription can be counteracted by USP4 gene knockdown; P+I is co-treated with PMA and Inomycin for simulating TCR signaling pathway stimulation, PMA translation is phorbol ester, and ionomycin is Ionomycin;

Figure D. FLAG-RORγt-Jurkat cells were added (P/I) overnight, cells were harvested by adding TGFβ (1 ng/ml) and IL-6 (20 ng/ml) or simultaneously with USP4 inhibitor for 8 h, and then lysed. Using FLAG for co-immunoprecipitation, and then using endogenous ubiquitin antibody and other corresponding antibodies for immunoblot analysis, the results showed that the reduction of RORγt ubiquitination by TGFβ and IL-6 can be inhibited by the activity of USP4. Agent offset; ubi is the abbreviation of ubiquitin protein;

Figure E, F. FLAG-RORγt-Jurkat cells were added (P/I) overnight, stimulated with different treatments of TGFβ (1 or 5 ng/ml) and IL-6 (20 ng/ml) for 8 h. The cells were harvested and fixed with paraformaldehyde. After 30 min, 1% BSA was blocked for 1 h and then incubated with USP4 antibody for 1 h. After washing three times with PBS, the fluorescent secondary antibody (red) was incubated, DAPI (blue) was incubated for 1 h, and PBS was washed three times for immunofluorescence experiments. The bar graph is used to represent the nuclear shuttle ratio of USP4 by the value of blue light (indicating the nucleus) compared with red light (representing the nuclear and cytoplasmic USP4). The results show that the effects of TGFβ and IL-6 on RORγt play a role in affecting the nuclear localization of USP4 (in the figure, it can be seen that a single high-concentration TGFβ or a single plus IL-6USP4 is relatively abundant, and TGFβ and IL-6USP4 are added simultaneously. The nuclear reduction increases the amount of USP4 in the nucleus and promotes the deubiquitinating enzyme into the nucleus.

Figure 5 shows that USP4 inhibitors can impair Th17 cell differentiation and are highly expressed in Th17-dominant rheumatic heart disease samples,

Figures A, B and C. People

T cells were sorted by BD FACS AriaII flow cytometry, and treated with DMSO or USP4 inhibitor (1 uM, 2 uM, inhibitor (USPi) for Vialinin A) for 7 days under Th17 induction conditions. -PCR and flow cytometric analysis by PE-RORγt, APC-FOXP3 and Percp/cy5.5-IL-17 antibodies, the results showed that the addition of USP4 inhibitors during the initial phase of Th17 differentiation and the whole process can be obvious The inhibition of Th17 differentiation efficiency is due to a decrease in the stability of RORγt or an increase in the expression level of FOXP3 during differentiation.

Figure D. Enrichment of CD4 ⁺ T cells with magnetic beads in peripheral blood of rheumatic heart disease and healthy controls, followed by qRT-PCR analysis with specific primers, showing up-regulation of USP4 and IL in samples from patients with rheumatic heart disease The up-regulation of -17 has important correlations, explaining that under the inflammatory conditions of autoimmune diseases in vivo, USP4 may be used as a specific drug target for the treatment of diseases, and its specific chemical inhibitors can be used as new therapeutic drugs.

Figure 6 shows deubiquitinating enzymes USP2, USP4, USP12, USP14, USP39 enhancing RORγt-mediated transcriptional activity of the Il17a promoter, and USP2, USP4, USP17 are capable of effectively removing ubiquitination of RORγt, wherein

Figure A. Myc-USPs (USP with MYC tag expressing MYC tag and different USP genes, ie deubiquitinating enzyme protein), HA-RORγt, Il17a promoter and β-galactoside The enzyme reporter gene was co-transfected into human renal epithelial cells HEK 293T cells. After harvesting the cells 36 hours later, the effect of deubiquitinating enzyme on RORγt-mediated transcriptional activity was observed by luciferase reporter gene assay;

Figure B. Myc-DUBs (DUBs with MYC tags expressing MYC tags and different DUB genes, ie deubiquitinating enzyme proteins), FLAG-RORγt and HA-Ubiquitin (representing HA tags and The HA-tagged ubiquitin protein expressed by the ubiquitin gene was co-transfected into HEK 293T cells, and the cells were collected 48h later, lysed by RIPA buffer, and then co-immunoprecipitated with anti-Flag antibody, and then immunoblotted with the corresponding antibody. Detection

Figure C. Il17a promoter and β-galactosidase reporter gene, Myc-USP17 and Flag-RORγt co-transfected human renal epithelial cells HEK 293T cells, harvested cells after 36 hours, lysed cells with reporter lysate, and added reaction The effect of substrate detection related activity;

Panel D. The Il17a promoter and the Renilla reporter gene, Myc-USP17 were electrotransformed in the Flag-RORγt stable transfectant, and were incubated with PMA and Inomycin for 12 hours after 36-48 hours, and were sampled by dual luciferase reporter assay.

Figure 7 shows the USP17 and RORγt interactions,

Figure A. USP17 interacts with RORγt. Flag-RORγt and Myc-USP17 are co-transformed into HEK 293T cells in six-well plates. Cells are harvested 48 hours later and 1 μg of anti-Flag or anti-Myc monoclonal is added after lysis. The antibody was precipitated and subjected to immunoblot analysis by monoclonal antibodies against Myc or anti-Flag. The expression levels of RORγt and USP17 in cell lysates were also analyzed by immunoblotting;

Figure B. Naive T cells were induced for 7 days under Th17 induction conditions, and then the cells were harvested and lysed by RIPA buffer, then co-immunoprecipitated with USP17 antibody, and then immunoblotted with the corresponding antibody;

Figure C. Schematic diagram of the RORγt domain;

Panel D. USP17 can interact with two domains of RORyt. A total of Flag-tagged RORγt three truncated mutants and USP17 were transfected into 293T cells, cells were harvested 48 hours later, lysed, precipitated with anti-Myc monoclonal antibody, and immunoblotted with anti-Myc or anti-Flag monoclonal antibody. . The expression levels of RORγt mutant and USP17 in cell lysates were also analyzed by immunoblot.

Figure 8 shows USP17 stable RORγt, where

Figure A. USP17 enhances RORγt stability. The same dose of Flag-RORγt co-transfects human renal epithelial cells HEK 293T cells with increasing doses of Myc-USP17 and Myc-USP17C89S, and harvests cells 48 hours later. USP17 in cell lysates And RORγt expression levels were analyzed by immunoblotting;

Figure B. USP17 stabilizes RORγt and extends its half-life. Myc-USP17 and Flag-RORγt co-transform human renal epithelial cells HEK 293T cells, and treated with protein synthesis inhibitor CHX for a period of time (0h, 4h, 8h, 12h) before harvesting cells, USP17 and RORγt expression levels in cell lysates Immunoblot analysis.

Figure C. Quantitative analysis of the intensity of RORγt protein bands treated with different treatments at different times in 8B using Image J image analysis software.

Figure 9 shows the ubiquitination of USP17 to RORγt K48, where

Figure A. Myc-RORγt and Flag-Ubiquitin were co-transformed into 293T cells. After 48 hours, the cells were harvested and treated with pre-protein degradation inhibitor MG132 (20 um/ml) for 3 h, precipitated by anti-Myc monoclonal antibody, and then Monoclonal antibodies against Myc or anti-Flag were subjected to immunoblot analysis, and the expression level of RORγt in cell lysates was also analyzed by immunoblotting;

Figure B. Transfection of Flag-RORγt, Myc-USP17, Myc-USP17C89S and His-Ubiquitin in 293T cells, cells were collected 48 hours later, and MG132 (20 um/ml) was collected for 3 h before harvesting, using Ni-NTA nickel chelate The resin was purified and precipitated, and then subjected to immunoblot analysis by anti-Myc, anti-Flag and anti-His monoclonal antibodies. The expression levels of RORγt, ubiquitin and USP17 in the cell lysate were also analyzed by immunoblotting.

Figure C. USP17 is capable of ubiquitinating K48-related ubiquitin, 293T cells are transfected with Flag-RORγt, Myc-USP17 and His-Ubiquitin mutants K63only and K48only, cells are harvested 48 hours later, and pre-cell MG132 is collected ( 20 um/ml) treatment for 3 hours, purification of the precipitate using Ni-NTA nickel chelate resin, and immunoblot analysis by anti-Myc, anti-Flag and anti-His monoclonal antibodies, expression of RORγt, ubiquitin and USP17 in cell lysate Levels were also analyzed by immunoblotting.

Figure 10 shows that down-regulation of USP17 in Th17 cells reduces the protein level of RORγt and affects the transcription level of Th17-associated cytokines, wherein

Figure A. Specific shRNAs of Myc-USP17 and USP17 were simultaneously transduced in 293T cells, cells were harvested 48 hours later, and lysed and immunoblotted by anti-Myc monoclonal antibody to detect shRNA knockout efficiency;

Figure B. DR8.9, shRNA, VSVG three-plasmid system packaging lentivirus, collecting virus after 36-48 hours, infecting Flag-RORγt stably transfected cells, and constructing shRNA to carry puromycin resistance, so after one week of screening with puromycin, After cell lysis, the expression of related proteins was detected by immunoblotting with anti-RORγt and anti-USP17 antibodies.

Figure C. DR8.9, shRNA, VSVG three-plasmid system packaged lentivirus, collected virus after 36-48 hours, infected with Th17 cells differentiated in vitro, constructed by shRNA carrying puromycin resistance, so after one week of screening with puromycin, After cell lysis, the expression of related proteins was detected by immunoblotting with anti-RORγt antibody and anti-USP17 antibody.

Figure D. Purification of RNA by Th17 cells after one week of puromycin screening and inversion of cDNA, and the transcription levels of related genes (USP17, IL-17A, IL-17F, IL-23R) were detected by real-time quantitative PCR.

Figure 11 shows that USP17 transcription levels are significantly elevated in patients with systemic lupus erythematosus (SLE),

Figure A. Peripheral venous blood from patients with systemic lupus erythematosus diagnostic criteria and healthy controls. CD4 ⁺ T cell enrichment kit was used to isolate CD4 ⁺ T cells, extract RNA and reverse cDNA, and detect related genes by real-time quantitative PCR. ;

Figure B. Correlation analysis of USP17 and IL-17A and IL-17F transcription levels;

Figure C. Grouping patients according to the SLE patient disease activity index and analyzing changes in USP17 transcript levels in the active and inactive groups.

Figure 12 shows the therapeutic effect of Vialinin A on its own encephalomyelitis, wherein

Figure A shows the experimental flow chart. On day 0, mice were injected with pertussis toxin in the tail vein, and the MOG peptide was subcutaneously immunized to induce experimental autoencephalomyelitis. Vialinin A (inhibitor of USP4) was intraperitoneally injected on days 9, 12, and 15, respectively, and the same amount of solvent (PBS containing 5% DMSO) was injected into the control group. The disease status of the mice was observed, and the mice were sacrificed on the 25th day for experimental analysis.

Panel B shows the disease score. The results showed that the control group started on the 10th day, and then the disease developed rapidly and entered the plateau on the 19th day. There was no significant difference between the drug-administered group and the control group in the early stage of the disease. In the 18 to 21 days with severe disease, the drug-administered group was significantly better than the control group.

detailed description

Through extensive and intensive research, the present inventors have unexpectedly discovered that deubiquitinating enzymes have a significant effect on up-regulating the stability of RORγt, thereby upregulating the activity of T helper cells and their differentiation and up-regulating the helper T cell cytokines. Expression, on the basis of which the present invention has been completed.

the term

Deubiquitinating enzyme and preparation method thereof

Approximately 100 deubiquitinating enzymes have been discovered to date, the main function of which is to hydrolyze ubiquitin molecules from proteins linked to ubiquitin by hydrolyzing the carboxy terminal residues on ubiquitin. According to their structural and functional characteristics, they can be divided into five families: Ubiquitin C-terminal hydrolases (UCHs), Ubiquitin-specific proteases (USPs), and ovarian tumor-associated proteases. (Ovarian tumor, OTU), Ataxin-3 (containing Josephin domain), MPN (+)/JAMM protease (JAB1/MPN/Mov34 metlloenzyme domain zinc-dependent metalloprotease family).

It has been found that deubiquitinating enzyme functions include: 1 deubiquitinating enzyme activates ubiquitin proprotein by means of co-translation. Ubiquitin is expressed as a proprotein and fused to a ribosomal protein or linear polyubiquitin, and the polyubiquitin gene product must also remove excess residues at the carbon end to activate ubiquitin. 2 Deubiquitination can recover ubiquitin captured by small cell nucleophiles containing a thiol ester intermediate, thereby participating in ubiquitination of the protein. 3 Deubiquitinating enzyme reversely removes ubiquitination or ubiquitin-like modification of the target protein. The ubiquitination of the ubiquitinating enzyme antagonist protein is similar to the action of phosphatase in the kinase/phosphatase regulatory pathway. 4 Deubiquitinase is responsible for the regeneration of monoubiquitin from non-anchored polyubiquitin chains.

The deubiquitinating enzyme USP4 specifically removes the monomeric or polyubiquitination of the substrate protein and prevents it from entering the proteasome to degrade or affect its function, thereby affecting tumorigenesis, apoptosis, immune response and the like. The study found that USP4 can promote tumor development through target action and tumor suppressor gene P53, and can also promote tumor growth by stabilizing tumor growth factor receptor (TGFβR1). However, the impact of USP4 on the immune system remains unclear.

The deubiquitinating enzyme USP17 interacts with SDS through the HABM domain to inhibit tumor cell growth. It has recently been discovered that USP17 is capable of modulating viral-induced type I interferon signaling by deubiquitination of RIG-1 and MDA5. Deletion of USP17 prevents normal cytoskeletal rearrangement and chemokine-induced Rho GTPase membrane localization, therefore, USP17 is also essential for Rho GTPase localization during normal cell movement. In conclusion, the effect of USP17 on apoptosis, cell proliferation, and cell cycle progression is an important factor in maintaining cell homeostasis. Rheumatic heart disease is caused by infection with streptococcus, a component of streptococcal cell wall is similar to the connective tissue of the human heart valve, and then develops into a disease that destroys the heart valve, and systemic lupus erythematosus is also a common clinical disease. The autoimmune diseases, the clinical manifestations are complex and changeable, the disease is protracted and difficult to heal, and the current treatment strategies are still scarce. Current research indicates that the balance of helper Th17 and regulatory Treg is critical for the development and treatment of rheumatic heart disease.

Therefore, in-depth study of the biochemical activity, physiological functions and regulatory molecular mechanisms of helper T cells and transcription factors RORγt, IL-17A, IL-17F, IL-21 and IL-23R for therapeutic control of human body The immunological activity of helper T cells and immunotherapy based on helper T cells are essential. This provides new research propositions and challenges for translating basic immunization research into clinical research and understanding human-specific autoimmune diseases such as multiple sclerosis. In the study of the present invention, it was found that USP4 and USP17 were significantly up-regulated in the blood samples of these two types of patients, respectively, and it is expected to become a new therapeutic target.

The deubiquitinating enzyme of the present invention is selected from the group consisting of a wild-type or mutant deubiquitinating enzyme, and is also selected from: an active fragment of a deubiquitinating enzyme having the same function as a wild-type deubiquitinating enzyme. Or derivatization of derivatives of ubiquitinating enzymes.

In another preferred embodiment, the derivative of the deubiquitinating enzyme is selected from the group consisting of: a modified deubiquitinating enzyme molecule, an amino acid sequence homologous to a natural deubiquitinating enzyme, and having natural deubiquitinating enzyme activity Protein molecule, dimer or multimer of deubiquitinating enzyme, fusion protein containing deubiquitinating enzyme amino acid sequence.

As used herein, the deubiquitinating enzyme "USP4" or "USP17" refers to a protein which has (a) and (Wang L, et al. J Virol. 2013), (de la Vega M, et The "USP4" or "USP17" substantially identical amino acid sequences described in al. Nat Commun. 2011) and (b) have the same biological activity as the native "USP4" or "USP17" and have substantially the same amino acid sequence. "USP4" or "USP17" of the present invention includes, but is not limited to, human "USP4" or "USP17", recombinant human "USP4" or "USP17", murine "USP4" or "USP17" and/or recombinant mouse "USP4" "or "USP17".

In some embodiments of the invention, the nucleotide sequence of USP4 is as set forth in SEQ ID NO.

The amino acid sequence of USP4 is:

MAEGGGCRERPDAETQKSELGPLMRTTLQRGAQWYLIDSRWFKQWKKYVGFDSWDMYNVGEHNLFPGPIDNSGLFSDPESQTLKEHLIDELDYVLVPTEAWNKLLNWYGCVEGQQPIVRKVVEHGLFVKHCKVEVYLLELKLCENSDPTNVLSCHFSKADTIATIEKEMRKLFNIPAERETRLWNKYMSNTYEQLSKLDNTVQDAGLYQGQVLVIEPQNEDGTWPRQTLQSKSSTAPSRNFTTSPKSSASPYSSVSASLIANGDSTSTCGMHSSGVSRGGSGFSASYNCQEPPSSHIQPGLCGLGNLGNTCFMNSALQCLSNTAPLTDYFLKDEYEAEINRDNPLGMKGEIAEAYAELIKQMWSGRDAHVAPRMFKTQVGRFAPQFSGYQQQDSQELLAFLLDGLHEDLNRVKKKPYLELKDANGRPDAVVAKEAWENHRLRNDSVIVDTFHGLFKSTLVCPECAKVSVTFDPFCYLTLPLPLKKDRVMEVFLVPADPHCRPTQYRVTVPLMGAVSDLCEALSRLSGIAAENMVVADVYNHRFHKIFQMDEGLNHIMPRDDIFVYEVCSTSVDGSECVTLPVYFRERKSRPSSTSSASALYGQPLLLSVPKHKLTLESLYQAVCDRISRYVKQPLPDEFGSSPLEPGACNGSRNSCEGEDEEEMEHQEEGKEQLSETEGSGEDEPGNDPSETTQKKIKGQPCPKRLFTFSLVNSYGTADINSLAADGKLLKLNSRSTLAMDWDSETRRLYYDEQESEAYEKHVSMLQPQKKKKTTVALRDCIELFTTMETLGEHDPWYCPNCKKHQQATKKFDLWSLPKILVVHLKRFSYNRYWRDKLDTVVEFPIRGLNMSEFVCNLSARPYVYDLIAVSNHYGAMGVGHYTAYAKNKLNGKWYYFDDSNVSLASEDQIVTKAAYVLFYQRRDDEFYKTPSLSSSGSSDGGTRPSSSQQGFGDDEACSMDTN (SEQ ID NO.2)

In some embodiments of the invention, the nucleotide sequence of USP17 is set forth in SEQ ID NO.

The amino acid sequence of USP17 is:

MEDDSLYLGGEWQFNHFSKLTSSRPDAAFAEIQRTSLPEKSPLSCETRVDLCDDLAPVARQLAPREKLPLSSRRPAAVGAGLQNMGNTCYVNASLQCLTYTPPLANYMLSREHSQTCHRHKGCMLCTMQAHITRALHNPGHVIQPSQALAAGFHRGKQEDAHEFLMFTVDAMKKACLPGHKQVDHHSKDTTLIHQIFGGYWRSQIKCLHCHGISDTFDPYLDIALDIQAAQSVQQALEQLVKPEELNGENAYHCGVCLQRAPASKTLTLHTSAKVLILVLKRFSDVTGNKIAKNVQYPECLDMQPYMSQTNTGPLVYVLYAVLVHAGWSCHNGHYFSYVKAQEGQWYKMDDAEVTASSITSVLSQQAYVLFYIQKSEWERHSESVSRGREPRALGAEDTDRRATQGELKRDHPCLQAPELDEHLVERATQESTLDHWKFLQEQNKTKPEFNVRKVEGTLPPDVLVIHQSKYKCGMKNHHPEQQSSLLNLSSTTPTHQESMNTGTLASLRGRARRSKGKNKHSKRALLVCQ (SEQ ID NO.4)

The nucleotide sequence of USP2 is shown in SEQ ID NO.: 5.

The amino acid sequence of USP2 is:

MRTSYTVTLPEDPPAAPFPALAKELRPRSPLSPSLLLSTFVGLLLNKAKNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQRLYMRDLHHGSNAHTALVEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAPRFVGYNQQDAQEFLRFLLDGLHNEVNRVTLRPKSNPENLDHLPDDEKGRQMWRKYLEREDSRI GDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKRGYPEVTLMDCMRLFTKEDVLDGDEKPTCCRCRGRKRCIKKFSIQRFPKILVLHLKRFSESRIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSNHSGTTMGGHYTAYCRSPGTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYELASPPSRM(SEQ ID NO.:6)

The term "substantially identical amino acid sequence" refers to a difference in sequence or caused by one or more amino acid changes (deletion, addition, substitution), but such alteration does not substantially reduce its biological activity. Any deubiquitinating enzyme that meets the "substantially identical" requirement is included in the present invention, whether it is glycosylated (ie, derived from a natural or eukaryotic expression system) or non-glycosylated ( That is, derived from prokaryotic expression systems or chemically synthesized).

"Deubiquitinating enzymes" also include PEGylated deubiquitinating enzymes as well as covalently modified deubiquitinating enzyme proteins. For example, various activated polyethylene glycols (PEG) having a molecular weight of 5,000 to 100,000 can be used to polymerize IL-17 to prolong its half-life. For specific procedures, see Greenwald et al., Bioorg. Med. Chem. Lett. 1994, 4, 2465; Calettiti et al., IL Farmaco, 1993, 48, 919; Zalipsky and Lee, Polyethylene glycol chemistry: Biotechnology and biology Medical Applications, edited by JM Harris, Plenum Press, NY, 1992.

The deubiquitinating enzyme of the present invention can be cloned and expressed by genetic recombination techniques. Host cells for expression include prokaryotic cells, yeast cells, or higher eukaryotic cells. In addition to prokaryotic cells, eukaryotic cells such as filamentous fungi or yeast are equally suitable for expressing or cloning the interleukin-17 of the present invention. The host cell of the deubiquitinating enzyme of the present invention for expressing glycosylation is derived from a multicellular organism. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, plant cells. Examples of suitable mammalian host cells include Chinese hamster ovary cells (CHO), COS cells. One of ordinary skill in the art will know how to select a suitable host cell.

The above host cells are transfected or transformed by a deubiquitinating enzyme expression vector or a cloning vector and cultured in a conventional nutrient medium, which is modified to be suitable for inducing a promoter and selectivity. A transforming transformant or amplifying a deubiquitinating enzyme-encoding gene sequence. Selection of culture conditions such as medium, temperature, pH, etc. will be known to those of ordinary skill in the art. General principles, protocols, and techniques for how to maximize cell culture fertility can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

The deubiquitinating enzyme of the present invention can be directly expressed not only by genetic recombination, but also by forming a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence located at the N-terminus of the mature protein or polypeptide, or may be located in the mature protein. Or other polypeptide fragments having a specific cleavage site at the N-terminus of the polypeptide.

Deubiquitinating enzyme dimer and preparation method thereof

The DNA sequence encoding the deubiquitinating enzyme dimer or fusion protein of the present invention can be fully synthesized. The DNA sequence encoding the de-ubiquitinating enzyme first monomer and/or the deubiquitinating enzyme second monomer can also be obtained by PCR amplification or synthesis, and then spliced together to form a fusion protein encoding the present invention. DNA sequence.

In order to increase the expression level of the host cell, the deubiquitinating enzyme dimer coding sequence can be engineered, for example, using a host cell-preferred codon to eliminate sequences that are not conducive to gene transcription and translation. In the present invention, the yeast cell or mammalian cell-preferred codon can be used, and the deubiquitinating enzyme dimer gene can be detected by computer DNA software, and the sequence which is not conducive to gene transcription and translation in the gene is excluded, including Intron cleavage site, transcription termination sequence, and the like.

After obtaining the DNA sequence encoding the novel fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by isolation and purification.

The term "vector," as used herein, includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors, and the like.

In the present invention, various carriers known in the art such as commercially available carriers can be used. For example, a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and is involved in the secretion of a polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for secretory leader sequences means adjacent in the reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

After obtaining the transformed host cell, the cell can be cultured under conditions suitable for expression of the fusion protein of the present invention to express the fusion protein. The expressed fusion protein is then isolated.

RORγt

The orphan nuclear receptor RORgammat (RORγt) is important for the differentiation and function of helper Th17, and is a specific transcriptional regulator of cell differentiation. It can also regulate Th17 inflammatory response by inducing IL-17 secretion. The presence of an intermediate Foxp3 ⁺ RORγt ⁺ cell was also found in other helper T cells, and this intermediate appeared in the NOD mouse T cell before inflammation and could be expanded without differentiation, so It is speculated that it may be a temporary cell population before differentiation into Treg/Th17. The direction in which this temporary cell population is differentiated may depend on the cellular microenvironment in which it is located.

The term "treating" refers to the administration of interleukin-17 of the present invention to a subject in need of treatment for the purpose of curing, alleviating, ameliorating, alleviating, affecting the disease, symptoms, and disease predisposition of the subject.

The term "therapeutic subject" refers to rats, humans, and other mammals.

The term "therapeutically effective amount" refers to an amount of a deubiquitinating enzyme or derivative thereof, a deubiquitinating enzyme agonist or antagonist capable of achieving therapeutic purposes in a subject. One of ordinary skill in the art will appreciate that the "therapeutically effective amount" may follow the route of administration of the ubiquitinating enzyme or derivative thereof, the deubiquitinating enzyme agonist or antagonist, the pharmaceutical excipients used, and other drugs. The combination of medications varies.

Pharmaceutical composition and method of administration

The pharmaceutical composition of the present invention comprises a deubiquitinating enzyme of the present invention or a derivative thereof, a deubiquitinating enzyme agonist or antagonist (active ingredient), and a pharmaceutically acceptable excipient or a safe or effective amount Carrier. By "safe, effective amount" it is meant that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical compositions contain from 0.001 to 1000 mg of active ingredient per dose, preferably from 0.05 to 300 mg of active ingredient per dose, more preferably from 0.5 to 200 mg of active ingredient per dose.

The active ingredient of the present invention and a pharmaceutically acceptable salt thereof can be formulated into various preparations containing the active ingredient of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount and a pharmacologically acceptable form. Agent or carrier. By "safe, effective amount" it is meant that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the active ingredient is determined according to the specific conditions such as the age, condition, and course of treatment of the subject.

"Pharmacologically acceptable excipient or carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity . By "compatibility" it is meant herein that the components of the composition are capable of intermixing with the compounds of the invention and with each other without significantly reducing the potency of the compound. Examples of pharmaceutically acceptable excipients or carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants ( Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.

When the composition of the present invention is administered, it can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously) or topically.

The compositions of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

The microcapsules containing the composition of the present invention can be used for sustained release administration of the active ingredient of the present invention. The microcapsule sustained release drug delivery technology of recombinant protein has been successfully applied to recombinant human growth hormone (rhGH), recombinant human interferon (rhIFN), interleukin-2 and MNrgp120 (Johnson et al., Nat. Med., 2:795- 799 (1996); Yasuda, Biomed. Ther 27: 1221-1223 (1993); WO 97/03692, WO 96/40072, WO 96/07399; US Pat. No. 5,654,010.

The sustained release preparation of the active ingredient of the present invention can be prepared using a lactic acid glycolic acid high polymer (PLGA) having good biocompatibility and broad biodegradability. The degradation products of PLGA, lactic acid and glycolic acid can be quickly eliminated by the human body. Moreover, the degradation ability of the polymer can be extended from several months to several years depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents form lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41)).

When a pharmaceutical composition is used, a safe and effective amount of the active ingredient of the present invention is applied to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage for a 60 kg body weight. The dose per administration is usually 0.01 to 300 mg, preferably 0.5 to 100 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

In a preferred embodiment of the invention, Vialinin A (an inhibitor of USP4) is administered at a dose of from 1 to 100 ng/kg body weight, preferably from 5 to 50 ng/kg body weight, more preferably 10 ng/kg body weight.

One of the main objects of the present invention is to provide ubiquitination pathway-associated factors and their agonists or antagonists in the regulation of RORγt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activities. use. Another object of the present invention is to provide use of these regulatory factors for regulating the transcriptional regulatory activity of RORγt on its target gene, thereby regulating the immunosuppressive activity of RORγt ⁺ Th17.

In one aspect of the invention there is provided a composition for modulating RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activity in a ubiquitination pathway-associated factor, agonist or antagonist thereof Use of the ubiquitination pathway-related factor selected from: USP4/USP17, a signal factor that induces USP4/USP17 expression or alters its nuclear localization, and/or their coding sequence; and/or the ubiquitin An agonist or antagonist of a pathway-associated factor.

In another embodiment of the present invention, the regulation of RORγt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R, and IL-2 activity may be positive or negative regulation, wherein the ubiquitin Pathway-associated factors or agonists thereof are used to positively regulate RORγt, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activities and negatively regulate FOXP3 activity, antagonism of ubiquitination pathway-associated factors The agents are used to negatively regulate RORyt, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activities and positively regulate FOXP3 activity.

In a preferred embodiment, the antagonist is shUSP4, shUSP17.

In another preferred embodiment, the antagonist is a USP4 inhibitor (4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-diyl ester benzeneacetic acid).

In another embodiment of the invention, the pro-inflammatory cytokine is selected from the group consisting of IL-17 and IL-6.

In a preferred embodiment of the invention, the use is a use of a ubiquitination pathway-related factor in the preparation of a composition that positively modulates RORγt activity, wherein the ubiquitination pathway-related factor is selected from the group consisting of: USP4/USP17, induction USP4/USP17 expresses or alters its nuclear factor-localized signaling factor, and/or an agonist or antagonist of the ubiquitination pathway-associated factor.

In a preferred embodiment of the invention, the activity disorder is hyperactivity or activity is too low.

In another embodiment of the invention, the correlation is related to RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activity. USP4/USP17 protein-specific shRNA can inhibit IL-17A, IL-17F, IL-21, IL-23R and IL-2 mRNA expression and promote FOXP3 mRNA expression.

In a preferred embodiment of the invention, the composition further comprises a pharmaceutically, hygienic or immunologically acceptable carrier.

In another aspect of the invention, there is provided a composition for modulating the activity of RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 comprising:

(a) one or more ubiquitination pathway-related factors selected from the group consisting of USP4/USP17, a signal factor that originally induces USP4/USP17 expression or alters its nuclear localization, and/or their coding sequence; and/ Or an agonist or antagonist of the ubiquitination pathway-related factor.

(b) A pharmaceutically, immunologically or health-care acceptable carrier.

In a preferred embodiment, the composition is for the treatment or prevention of dysregulation of activity with RORγt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 (ie, activity is too high or activity is too low) a related disease or condition or as a vaccine adjuvant.

In a preferred embodiment, the composition further comprises one or more additional active substances that modulate RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2.

In another preferred embodiment, the proto-oncogene protein USP4/USP17 reduces ubiquitination associated with RORyt degradation.

In a preferred embodiment, the content of the conditioning factor in the composition is from 0.05 to 99.5% by weight, preferably from 0.1 to 95% by weight, more preferably from 1 to 90% by weight, still more preferably from 5 to 80% by weight.

In another preferred embodiment, the composition is an injection, a tablet, a granule, a powder or a capsule.

In a further aspect of the invention, there is provided a method of modulating the activity of RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2, the method comprising administering one or more a ubiquitination pathway-related factor selected from the group consisting of USP4/USP17, a signal factor that induces USP4/USP17 expression or alters its nuclear localization, and/or their coding sequence; and/or the ubiquitination pathway-associated factor An agonist or antagonist.

In another preferred embodiment, the present invention still further provides a method of treating or preventing a disease or condition associated with dysregulation of RORyt, FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2 activity, The method comprises administering to a subject in need of such treatment or prevention an effective amount of one or more ubiquitination pathway-related factors selected from the group consisting of USP4/USP17, a signal that induces USP4/USP17 expression or alters its nuclear localization. Factores, and/or their coding sequences; and/or agonists or antagonists of the ubiquitination pathway-related factors.

In another preferred embodiment, the method is further for increasing the immunogenicity of the vaccine.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

The main advantages of the invention are:

(1) The role of deubiquitinating enzyme in regulating the stability of RORγt was first revealed;

(2) It was first revealed that IL-6 can be used to promote deubiquitinating enzymes from the cytoplasm into the nucleus.

(3) Deubiquitinating enzyme can enhance the stability of RORγt and thereby increase the activity and differentiation of helper T cells.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and changes to the present invention, and such modifications and variations are within the scope of the present invention.

The experimental methods in the following examples which do not specify the specific conditions can be carried out by conventional methods in the art, for example, refer to "Molecular Cloning Experimental Guide" (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor) Laboratory Press, 1989) or in accordance with the conditions recommended by the supplier. The sequencing method of DNA is a routine method in the art, and can also be provided by a commercial company for testing. The experimental materials used in the present invention are all available from commercially available sources.

Percentages and parts are by weight unless otherwise stated. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only.

Materials and Methods

1. Plasmids and vectors:

N-terminal FLAG-tagged RORγt, HA-tagged USP4, Myc-tagged USP17 and other plasmids and lentiviral packaging vector plasmids (PLKO.1, del8.9 and VSV-G) were cloned (see reference Liu, X.). ,Li,H.,Zhong,B.,Blonska,M.,Gorjestani,S.,Yan,M.,Tian,Q.,Zhang,DE,Lin,X.,and Dong,C.(2013)USP18inhibits NF -kappaB and NFAT activation during Th17differentiation by deubiquitinating the TAK1-TAB1 complex. The Journal of experimental medicine 210, 1575-1590).

USP4, USP17 gene was amplified from human peripheral blood mononuclear cell cDNA library. The primers were designed according to the sequence downloaded by Genbank as follows:

2. Antibody:

Flag antibody (M2) was purchased from Sigma. RORγt antibody was purchased from eBioscience. USP4, USP17 antibody was purchased from Sigma. His antibody was purchased from SAB. HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Promega.

3. Cells and their treatment:

Human HEK293T (purchased from the Chinese Academy of Sciences Cell Bank (catalog number: GNHu17)) in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, 100 units/ml of streptomycin at 37 ° C, 5% CO ₂ to cultivate.

FLAG-RORγt–Jurkat T cells (RORγt stably expressed lymphocyte strains, produced and preserved by our laboratory) contain 10% FBS, 100 units/ml penicillin, 100 units/ml non-essential amino acids and 100 units/ In 1640 medium of sodium pyruvate, the cells were cultured at 37 ° C under 5% CO ₂ .

Cell transfection: HEK293T was transfected with polyethyleneimine PEI (Polyethylenimine) (according to the operation) The book is carried out), and the sample is analyzed after 48 hours of transfection. Jurkat T was transfected with an electric shock method, see the electric shock method for Jurkat conditions.

The human embryonic kidney cell line HEK293T was co-transfected with PLKO.1, dR8.9 and VSV-G, and the cell culture supernatant was collected 48 and 72 hours after transfection.

The shUSP primer sequence is designed as follows:

The shCK primer sequence is as follows:

The synthesized shRNA primer sequence was annealed and ligated into the shRNA lentiviral expression vector pLKO.1 (purchased from Addgene). After mixing in the ratio of pLKO.1:dR8.9:VSVG=5:4:3, the cells were transferred to HEK 293T cells by calcium phosphate transfection, and the virus was collected 48 hours later. The virus suspension was centrifuged at low speed and filtered. Directly used for infection of the target cells.

4. Separation of reagents and nuclei and chromatin subfractions:

The USP4 inhibitor 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-diphenyl phenylacetic acid was purchased from R&D. TGFβ, IL6, IL-23 and IL-1β factors were purchased from Apotech. MG132 was purchased from Merck. Protein AG beads (Protein AG-beads) were purchased from Shanghai Yueke Biotechnology Co., Ltd.

5. Immunoprecipitation:

RIPA buffer (20 mM Tris/HCL pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-DOC, 1 mM EDTA and protease inhibitor 1 mM PMSF, 1×Cocktail, phosphatase inhibitor 1 mM Na ₃ VO ₄ , 1 mM NaF) lysed the cells. The cell lysate was first incubated with primary antibody for 1 hour, and then agarose beads conjugated with Protein A/G were added for 1 hour. After three washes with RIPA buffer, the bound proteins were detected by SDS-PAGE.

6. Immunoblotting:

The protein sample was electrophoresed by SDS-PAGE protein, transferred to a nitrocellulose membrane, blocked with 5% skim milk powder TBST for one hour, added with primary antibody for one hour, and then HRP-conjugated secondary antibody was incubated for one hour. The ECL substrate is exposed to color.

7. Gene site-directed mutagenesis:

USP4 mutant C331A, USP17 mutant C89S was obtained by mutant PCR (see references Zhu, S., Pan, W., Shi, P., Gao, H., Zhao, F., Song, X., Liu, Y). .,Zhao,L.,Li,X.,Shi,Y.,and Qian,Y.(2010)Modulation of experimental autoimmune encephalomyelitis through TRAF3-mediated suppression of interleukin 17receptor signaling.The Journal of experimental medicine 207,2647-2662 ).

8. His-pull down experiment:

This experiment is referred to by van Loosdregt, J., Fleskens, V., Fu, J., Brenkman, AB, Bekker, CP, Pals, CE, Meerding, J., Berkers, CR, Barbi, J., Grone, A. ,Sijts,AJ,Maurice,MM,Kalkhoven,E.,Prakken,BJ,Ovaa,H.,Pan,F.,Zaiss,DM,and Coffer,PJ(2013)Stabilization of the transcription factor Foxp3by the deubiquitinase USP7increases Treg- Cell-suppressive capacity. Immunity 39, 259-271.

9. Helper T cell subsets and induced Treg induction experiments:

This experiment is referred to by van Loosdregt, J., Fleskens, V., Fu, J., Brenkman, AB, Bekker, CP, Pals, CE, Meerding, J., Berkers, CR, Barbi, J., Grone, A. ,Sijts,AJ,Maurice,MM,Kalkhoven,E.,Prakken,BJ,Ovaa,H.,Pan,F.,Zaiss,DM,and Coffer,PJ(2013)Stabilization of the transcription factor Foxp3by the deubiquitinase USP7 increases Treg -cell-suppressive capacity.Immunity 39,259-271.

Example 1. USP4 is highly expressed in Th17 and affects the stability of its important transcription factor RORγt.

CD45RA+CD4+naive T cells were sorted from human peripheral blood cells (PBMC) by flow cytometry (FACS), and different helper T cell subsets and inducible regulation were obtained under different helper T cell induction conditions. Sex T cells (iTreg), extract RNA for specific primer real-time fluorescent quantitative PCR (q-RTPCR) or with specific antibody Western blotting (WB). (Results As shown in Fig. 1A, B, it can be seen from Fig. 1A that the transcription level of USP4 is highest in Th17 cells, and it can be seen from Fig. 1B that immunoblotting detection using anti-USP4 antibody, USP4 expression in Th17 cells Significantly improved, the results showed that USP4 was highly expressed in Th17 cells.

Naive T cells were induced for 7 days under Th17 induction conditions, and incubated with USP4 gene knockdown virus (puromycin resistance) overnight. After transfusion, 72 hours after infection, puromycin was added for one week, and cells were collected for q- RTPCR and immunoblot detection with anti-USP4 and anti-RORγt antibodies. The results are shown in Fig. 1C and D. It can be seen from Fig. 1C that the transcription level of Th17 cell-associated important cytokines is significantly reduced after knocking down USP4 (IL-17A, IL-17F, IL-2, IL-21, IL- 23R was down-regulated, but FOXP3 was up-regulated, and the transcription level of RORγt did not change; it can be seen from Figure 1D that the protein level of RORγt in Th17 cells was significantly decreased after knocking down USP4 by two different shUSP4.

The above results show that USP4 affects the stability of the important transcription factor RORγt of Th17 cells. Increasing the content of USP4 in cells can enhance the stability of RORγt, while the stability of RORγt is significantly decreased after the decrease of USP4 content, and affects the cytokines of Th17 cells. expression.

The above experimental results show that the deubiquitinating enzyme USP4 is highly expressed in Th17 and affects the stability of its important transcription factor RORγt.

Example 2. Effect of deubiquitinating enzyme USP4 on RORγt

The naive T cells were induced for 7 days under Th17 induction conditions, and then the cells were collected and lysed by RIPA buffer, and then co-immunoprecipitated with USP4 antibody, and then immunoblotted with anti-USP4 and anti-RORγt antibodies. As a result, as shown in Fig. 2A, endogenous USP4 was able to interact with RORγt in Th17 cells, and the results showed that USP4 could interact with RORγt.

HA-USP4 or its enzyme-activated mutant Ser311A mutant was co-transfected into HEK293T cells with FLAG-RORγt. After 48 hours, the protein synthesis inhibitor imine cyclohexanone (CHX) was treated in a time-sharing period, and the cells were harvested and lysed with HA and FLAG and other corresponding antibodies were used for immunoblot analysis. As a result, as shown in Fig. 2B, with the prolongation of time, the cells RORγt co-transduced with USP4 and RORγt hardly attenuated, and the cell stability of the single- or co-transformed enzyme-activated mutant was significantly decreased, and the results showed that: HA- USP4 blocks the attenuation of the FLAG-RORγ protein.

HA-USP4 and FLAG-RORγt and His-ubiquitin or its mutant (Lys48only, Lys63only) were co-transfected into HEK 293T cells, cells were harvested 48h and mutated with 8M urea and then labeled with nickel beads (beads) His (ie His-pull down assay) was combined with FLAG and other corresponding antibodies for immunoblot analysis. As a result, as shown in Fig. 2C, USP4 can remove the ubiquitination modification associated with RORγtK48, while the K63-related ubiquitination modification does not change. The results show that USP4 can remove the ubiquitination modification associated with degradation of specific RORγt.

FLAG-RORγt-Jurkat stable strain was stimulated with USP4 inhibitor (20 nM) for 4 hours, and non-stimulated and FLAG-Jurkat control, cells were collected and lysed, then co-immunoprecipitated with FLAG, and then ubiquitin was used. Antibodies and anti-USP4 and anti-FLAG antibodies were used for immunoblot analysis. As a result, as shown in Fig. 2D, USP4 can remove endogenous ubiquitination of specific RORγt. The results show that USP4 can remove endogenous ubiquitination modification of specific RORγt, and this process is USP4-dependent.

The above experimental results show that the deubiquitinating enzyme USP4 can bind to RORγt and stabilize the protein level of RORγt by specifically removing RORγt and degradation-related ubiquitination modification.

Example 3. USP4 promotes RORγt-induced transcription and production of interleukin-17

The IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) and different gradients of HA-USP4 and FLAG-RORγt were co-transfected into HEK 293T cells, and the luciferase reporter gene was collected at 48 h. After the lysate was lysed, the supernatant was added to the reaction solution and the blocking solution, and the fluorescence value was measured, and then HA and FLAG and other corresponding antibodies were used for immunoblot analysis. The results are shown in Fig. 3A. In the figure, the abscissa is the cell treated with different treatment, and the ordinate is the intensity of luciferase activity. It can be seen from the figure that USP4 promotes RORγt-induced interleukin-17A transcription in a dose-dependent manner. The results show that USP4 can promote RORγt-induced transcription and production of interleukin-17A.

HEK 293T cells were transfected into USP4 gene knockdown plasmid (puromycin resistance), transfected and transfected for 72 hours after transfection for one week. Cells were passaged and transferred to IL-17A luciferase promoter reporter gene ( -600-0bp), renilla (control) and FLAG-RORγt, cells were harvested 48 hours later for fluorescence measurement and immunoblot detection with the corresponding antibodies. The results are shown in Fig. 3B, C. It can be seen in Fig. 3B that RORγt induces down-regulation of interleukin-17A transcription after knocking down USP4, and the electrophoresis results in Fig. 3C show that the protein expression of RORγt is also significantly reduced after knocking down USP4. . The above results show that USP4 is indispensable for promoting RORγt-induced transcription and production of interleukin-17.

IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) were transferred into FLAG-RORγt-Jurkat or FLAG-Jurkat cells, and PMA and inomycin (myosin) were added after 44h. /I), after 4 hours, the cells were collected and lysed with luciferase reporter lysate, and the supernatant was added to the reaction solution and the blocking solution to measure the fluorescence value, and then immunoblot analysis was performed using USP4 and FLAG and other corresponding antibodies.

The results are shown in Figure 3D, E. Figure 3D shows that after activation of the TCR signal, USP4 can promote RORγt-induced interleukin-17 transcription, while USP4 inhibitors can inhibit this effect; Figure 3E shows that USP4 is able to stabilize RORγt and incorporate USP4 inhibition. The level of RORγt protein decreased after the agent. The result shows: in T cells Under the action of TCR signaling pathway, USP4 can promote the transcription and production of interleukin-17 induced by RORγt.

The above results indicate that USP4 promotes the transcription and production of interleukin-17 induced by RORγt in the exogenous system or in the TCR signaling pathway in T cells.

Example 4. IL-6 promotes RORγt-induced interleukin-17 by altering the nuclear localization of USP4 Transcription and production.

The IL-17A luciferase promoter reporter gene (-600-0bp) and renilla (control) were co-transfected into FLAG-RORγt-Jurkat or FLAG-Jurkat cells, and 36h was replaced with fresh culture solution (P/I) overnight. After adding TGFβ (1 ng/ml) and IL-6 (20 ng/ml) for 8 hours, the cells were collected and lysed with luciferase reporter lysate. The supernatant was added to the reaction solution and the blocking solution was added to measure the fluorescence value, and then USP4 was used. Immunoblot analysis was performed with FLAG and other corresponding antibodies. The results are shown in Figure 4A. It can be seen from the figure that IL-6 enhances the transcription of interleukin-17A induced by RORyt by USP4. The results show that IL-6 enhances the promotion of RORγt-induced interleukin-17A transcription by USP4.

FLAG-RORγt-Jurkat or FLAG-Jurkat cells were incubated with USP4 gene knockdown virus (puromycin resistance) overnight. After transfusion, 72 days after infection, add puromycin for one week, then transfer to IL-17A for luciferase initiation. Sub-reporter gene (-600-0bp), renilla (control), after 36h, fresh liquid was added to (P/I) overnight, TGFβ (1ng/ml) and IL-6 (20ng/ml) were added to stimulate the cells for 8h. The luciferase reporter lysate was lysed, and the supernatant was added to the reaction solution and the blocking solution, and the fluorescence was measured, and then subjected to immunoblotting using USP4 and FLAG and other corresponding antibodies. The results are shown in Fig. 4B, C, and D. It can be seen from Fig. B that after knocking down USP4, IL-6 stimulation does not enhance RORγt-induced interleukin-17A transcription. From Fig. C, it can be seen that knocking down USP4 The expression of RORγt protein was decreased in the post-stable strain. As can be seen from panel D, the decrease in the level of RORγt ubiquitination by TGFβ and IL-6 can be offset by the inhibitor of the activity of USP4. The results show that IL-6 enhances RORγt-induced interleukin-17 transcription and is USP4-dependent.

FLAG-RORγt-Jurkat cells were added (P/I) overnight, stimulated with different treatments of TGFβ (1 or 5 ng/ml) and IL-6 (20 ng/ml) for 8 h. The cells were harvested with paraformaldehyde for 30 min, 1%. After blocking for 1 h, BSA was incubated with the corresponding antibody of USP4 for 1 h, washed with PBS three times, then incubated with fluorescent secondary antibody (red), DAPI (blue) for 1 h, and washed with PBS three times for immunofluorescence experiments. The results are shown in Fig. 4E, F. It can be seen from the figure that the stimulation of TGFβ and IL-6 affects the localization of USP4 in the nucleus. This result shows that the effect of TGFβ and IL-6 on RORγt plays a role by affecting the nuclear localization of USP4.

The above experimental results indicate that IL-6 promotes RORγt-induced interleukin-17 transcription and production by altering the nuclear localization of USP4.

Example 5. USP4 inhibitors can impair Th17 cell differentiation and a rheumatoid heart dominated by Th17 High expression in dirty samples.

people

T cells were sorted by BD FACS AriaII flow cytometry, and treated with DMSO or USP4 inhibitor (1 uM, 2 uM, inhibitor Vial inin A) for 7 days under Th17 induction conditions. The cells were collected and subjected to qRT-PCR. Flow cytometric analysis was performed after staining with PE-RORγt and Percp/cy5.5-IL-17 antibodies. The results are shown in Fig. 5A, B. It can be seen from Fig. B that the transcription level of Th17-related transcription factor RORγt and cytokines such as IL-17A and IL-17F are significantly reduced after treatment with USP4 inhibitor, as can be seen from Fig. C. The expression of RORγt protein in Th17 cells was increased by flow cytometry, while the expression of Foxp3 was decreased. The results show that USP4 inhibitors can impair Th17 cell differentiation.

CD4+ T cells were enriched in the peripheral blood of rheumatic heart disease patients and healthy controls, and then subjected to qRT-PCR analysis using specific primers. The results are shown in Figure 5D. It can be seen from the figure that the transcription level of USP4 is significantly elevated in rheumatic heart disease samples. The results show that USP4 is highly expressed in Th17-dominant rheumatic heart disease samples.

The above results indicate that USP4 inhibitors can impair Th17 cell differentiation and are highly expressed in Th17-dominant rheumatic heart disease samples, which is expected to provide new strategies and targets for the treatment of rheumatic heart disease.

Example 6. Deubiquitinating enzymes USP2, USP4, USP12, USP14, USP39 enhance RORγt-mediated The transcriptional activity of the Il17a promoter, USP2, USP4, USP17 is effective in removing the ubiquitination of RORyt.

IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) and different gradients of USPs, enzyme deletion mutant C89S mutant and FLAG-RORγt co-transformed into HEK 293T cells, 48h cells were collected The luciferase reporter lysate was lysed and the supernatant was added to the reaction solution and the blocking solution to measure the fluorescence value. The results are shown in Figures 6A and 6C. From Fig. 6A, USP2, USP4, USP12, USP14, USP17 can be seen. , USP39 enhances the transcriptional activity of the Il17a promoter mediated by RORγt. It can be seen from Figure 6C that USP17 can promote RORγt-induced transcription of interleukin-17A. The results show that USP17 can promote RORγt-induced transcription and production of interleukin-17A.

Myc-DUBs, FLAG-RORγt and HA-Ubiquitin were co-transfected into HEK 293T cells. The cells were harvested for 48 hours, then lysed by RIPA buffer and then anti-Flag antibody was used for immunoprecipitation, and then the corresponding antibodies were used for immunoblotting. As shown in Fig. 6B, it can be seen from Fig. 6B that the addition of USP2 or USP4 or USP17 significantly reduces the level of ubiquitination of RORγt compared to the second lane without addition of deubiquitinating enzyme. USP2, USP4, USP17 can effectively Removal of ubiquitination of RORyt.

The results show that USP17 can most effectively remove the deubiquitination of RORγt, and USP2, USP4, etc. also have certain deubiquitination functions.

IL-17A luciferase promoter reporter gene (-600-0bp), renilla (control) were transferred into FLAG-RORγt-Jurkat cells, and PMA and inomycin (myomycin) (P/I) were added after 44h. After 4 hours, the cells were collected and lysed with luciferase reporter lysate, and the supernatant was added to the reaction solution and the blocking solution to measure the fluorescence value. The results are shown in Fig. 6D. It can be seen from the figure that USP17 is ROR after T cell activation. The enhanced effect of induced interleukin-17A transcription is dose dependent. The results show that USP17 can promote RORγt-induced transcription and production of interleukin-17A under the action of TCR signaling pathway in T cells.

The above results indicate that the deubiquitinating enzymes USP2, USP4, USP12, USP14, USP17, USP39 enhance RORγt-mediated transcriptional activity of the Il17a promoter under the action of the TCR signaling pathway in the T cell, USP2, USP4 , USP17 can effectively remove the ubiquitination of RORγt.

Example 7. USP17 binds to RORyt.

Myc-USP17 and FLAG-RORγt were co-transfected into HEK 293T cells. The cells were harvested 48h later and then lysed by RIPA buffer. The anti-Myc antibody or anti-Flag antibody was used for immunoprecipitation, and then the corresponding antibody was used for immunoblotting. As shown in Fig. 7A, it can be seen from the figure that USP17 and RORγt interact.

The naive T cells were induced under Th17 induction conditions for 7 days, then the cells were collected and lysed by RIPA buffer, then co-immunoprecipitated with anti-USP17 antibody, and then immunoblotted with the corresponding antibodies. The results are shown in Figure 7B. It can be seen that endogenous USP17 and RORγt interact in Th17 cells. This result shows that USP17 can interact with RORγt.

RORγt 1-75aa, 10-75aa and 248-497aa three-section truncation mutants and Myc-USP17 were overexpressed in HEK293T, immunoprecipitated with anti-Myc antibody, and finally detected by immunoblotting. The results are shown in Figure 7C. D, it can be seen from Figure C that RORγt contains two domains: a DNA binding domain and a ligand binding domain. It can be seen from Figure D that USP17 can interact with the two domains of RORγt. The results show that both domains of USP17 and RORγt can interact.

The above experimental results show that USP17 can interact with the two domains of RORγt.

Example 8. USP17 stabilizes RORyt.

Overexpression of Flag-RORγt and Myc-USP17 in HEK 293T cells, and USP17 transfection dose Increasingly, the corresponding antibody was used for immunoblotting, and the results are shown in Fig. 8A. It can be seen from the figure that the stability of USP17 to RORγt is dose-dependent. The results showed that USP17 had a dose-dependent effect on RORγt protein levels, while the enzyme-activated mutant USP17CS had no stabilizing effect.

After overexpressing RORγt and USP17 in the cells, the cells were treated with the protein synthesis inhibitor imine cyclohexanone (CHX) for 0h, 4h, 8h, 12h. The results are shown in Figure 8B, C, as seen in Figure B. USP17 can prolong the protein half-life of RORγt, and the stable effect of USP17 on RORγt can be seen quantitatively from Figure C. The results show that USP17 can effectively prolong the protein half-life of RORγt, and its enzyme-activated mutants have no similar function. The above results confirm the inference of USP17 to stabilize RORγt.

The above experimental results show that USP17 can stabilize the expression of RORγt protein and prolong its half-life.

Example 9. USP17 de-RORγt K48-related ubiquitination.

Myc-RORγt and FLAG-Ubiquitin were co-transfected into HEK 293T cells. The cells were harvested for 48 hours, then lysed by RIPA buffer and then co-immunoprecipitated with anti-Myc antibody, and then immunoblotted with the corresponding antibody. The results are shown in Figure 9A. It can be seen from the figure that RORγt can be ubiquitinated and ubiquitination can occur at both K48 and K63 positions. The results show that RORγt can be ubiquitinated and ubiquitination can occur at both K48 and K63 positions.

Flag-RORγt, Myc-USP17 and His-Ubiquitin were transiently expressed in HEK293T cells, and the precipitate was purified using Ni-NTA (nickel triacetate), and finally detected by immunoblotting. The results are shown in Fig. 9B. It can be seen that USP17 relies on its enzymatic activity to deubiquitinate RORγt. The results show that USP17 can deubiquitinate RORγt, while its enzyme-activated mutant (USP17C89S) has no function.

Flag-RORγt, Myc-USP17 and His-Ubiquitin or their mutants (K48only, K63only), the two mutants refer to ubiquitin itself except for the lysine at position 48 or 63, and the others are all mutated to arginine. The HEK 293T cells were co-transferred and purified by Ni-NTA (nickel triacetate). The experimental results are shown in Figure 9C. It can be seen from the figure that USP17 mainly mediates the deubiquitination of the RORγt K48 site. The results showed that USP17 mainly mediates deubiquitination of the RORγt K48 site.

The above experimental results show that the deubiquitinating enzyme USP17 mainly mediates the deubiquitination of the K48 position of the RORγt protein.

Example 10. Down-regulation of USP17 in Th17 cells reduces RORγt protein levels and affects Th17 correlation Transcriptional levels of cytokines.

USP17-specific short hairpin RNA (shUSP17-1, 2, 3) was cloned into the lentiviral vector pLKO.1 (purchased from Open Biosystems, USA), and the Myc-tagged USP17 and three specific shRNAs were transiently expressed, respectively. The shRNA silencing efficiency was examined by immunoblotting in HEK 293T cells, and the results are shown in Fig. 10A. It can be seen from the figure that shUSP17-3 has the best knockout efficiency. The results show that shUSP17-3 has the best gene silencing efficiency.

The sequence of shUSP17-1, 2, 3 is as follows

AAGCAGGAAGATGCCCATGAA (shUSP17-1, SEQ ID NO.: 14);

AAGTCACCACTCTCATGTGAG (shUSP17-2, SEQ ID NO.: 15);

GACACAGACAGGCGAGCAACG (shUSP17-3, SEQ ID NO.: 16).

The FLAG-RORγt-Jurkat stably transfected cells were incubated with USP17 gene silencing virus (puromycin antibody) overnight. After transfection, the cells were added to puromycin for 72 weeks after infection, and the cells were collected and immunoblotted with the corresponding antibodies. The results are shown in Fig. 10B. It can be seen from the figure that gene silencing of USP17 can reduce the expression of RORγt in stably transformed strains. The results showed that USP17 affects the stability of RORγt protein in FLAG-RORγt-Jurkat stably transfected cells.

Naive T cells were induced for 7 days under Th17 induction conditions, and incubated with USP17 gene silencing virus (puromycin resistance) overnight. After transfusion, 72 days after infection, puromycin was added for one week. Cells were harvested for q-RTPCR and immunoblotted with the corresponding antibodies. The results are shown in Figure 10C, D. It can be seen from Figure C that gene silencing of USP17 can reduce the expression of RORγt in Th17 cells. From Figure D, it can be seen that the transcription level of Th17-associated cytokine IL-17A after gene silencing USP17 Down. The results showed that USP17 affects the stability of the R17γ protein of Th17 cells and the important transcription factor RORγt.

The above experimental results indicate that deubiquitinating enzyme USP17 affects the stability of RORγt and its important transcription factors in Th17 cells.

Example 11. USP17 transcription levels were significantly elevated in patients with systemic lupus erythematosus.

In peripheral blood of patients with systemic lupus erythematosus and healthy controls, CD4 ⁺ T cells were enriched with magnetic columns, and then qRT-PCR analysis was performed using specific primers. The results are shown in Fig. 11A, B, and C. It can be seen from Fig. A that USP17, IL-17A, and IL-17F are up-regulated in patients with systemic lupus erythematosus; from Figure B, the transcription level of USP17 can be seen. IL-17A, IL-17F levels were positively correlated; it can be seen from Figure C that USP17 is significantly up-regulated in patients with active systemic lupus erythematosus. The results showed that USP17 was highly expressed in systemic lupus erythematosus and was significantly positively correlated with the levels of IL-17A and IL-17F.

The above experimental results show that USP17 is highly expressed in systemic lupus erythematosus samples and is expected to provide new strategies and target sites for its treatment.

Example 12. Deubiquitinating enzyme antagonists have significant therapeutic effects on autoencephalomyelitis.

On day 0, mice were injected with pertussis toxin in the tail vein, and the MOG peptide (purchased from Jill Biochemical (Shanghai) Co., Ltd.) was subcutaneously administered to induce experimental neoencephalomyelitis. Vialinin A (inhibitor of USP4) was intraperitoneally injected on days 9, 12, and 15, respectively, at a dose of 10 ng/kg of mouse body weight, and the control group was injected with the same amount of solvent (PBS containing 5% DMSO). The disease status of the mice was observed, and the mice were sacrificed on the 25th day for experimental analysis.

Figure 12 shows the therapeutic effect of Vialinin A on its own encephalomyelitis, wherein

Figure A shows the experimental flow;

Panel B shows the disease score. The results showed that the control group started on the 10th day, and then the disease developed rapidly and entered the plateau on the 19th day. There was no significant difference between the drug-administered group and the control group in the early stage of the disease. In the 18 to 21 days with severe disease, the drug-administered group was significantly better than the control group;

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

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Claims (15)

1. Use of a deubiquitinating enzyme or a deubiquitinating enzyme agonist, characterized in that it is used for the preparation of a preparation or kit for:

   (1) increasing the stability of RORγt; and/or

   (2) up-regulating helper T cell activity or promoting differentiation of helper T cells; and/or

   (3) modulating the expression or activity of a helper T cell cytokine; and/or

   (4) Treating and/or preventing diseases associated with hypoxia of helper T cells.
2. The use according to claim 1, wherein said helper T cell activity is too low means that helper T cell pro-inflammatory function is too low; and/or

   The disease associated with hypoxia of helper T cells is selected from the group consisting of tumors and infectious diseases.
3. The use according to claim 1, wherein the deubiquitinating enzyme is selected from one or more of the group consisting of: Ubiquitin-specific proteases (USPs) and the OUT family; preferably, the The ubiquitinating enzyme is selected from one or more of the following groups: USP2, USP3, USP4, USP5, USP7, USP10, USP12, USP14, USP17, USP18, USP21, USP22, USP30, USP39, USP44, YOD1, CYLD, and A20; More preferably, the deubiquitinating enzyme is USP2, USP4, USP17 or a combination thereof.
4. The use according to claim 1, wherein the helper T cell is selected from one or more of the group consisting of Th1, Th2, Th3, Th9, Th17 and Tfh; and/or

   The helper T cell cytokine is selected from one or more of the group consisting of FOXP3, IL-17A, IL-17F, IL-21, IL-23R and IL-2.
5. Use of a deubiquitinating enzyme antagonist, characterized in that it is used for the preparation of a preparation or kit for:

   (1) reducing the stability of RORγt; and/or

   (2) down-regulating helper T cell activity or inhibiting differentiation of helper T cells; and/or

   (3) modulating the expression or activity of a helper T cell cytokine; and/or

   (4) treating and/or preventing a disease associated with an excessively high level of helper T cell activity; and/or

   (5) up-regulating the expression of FOXP3, enhancing the immunosuppressive function of Treg; and/or

   (6) Treating and/or preventing diseases associated with regulatory T cell activity.
6. The use according to claim 5, characterized in that the disease associated with hyperactivity of helper T cells is selected from the group consisting of an inflammatory response and an autoimmune disease.
7. The use according to claim 5, wherein the deubiquitinating enzyme antagonist is selected from the group consisting of an antibody, a sh-RNA, a miRNA, an antisense oligonucleotide, a chemical inhibitor, or a combination thereof.
8. The use according to claim 7, wherein the chemical inhibitor is: 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2 ',3'-Diester phenylacetic acid (Vialinin A).
9. A composition for increasing the stability of RORyt, characterized in that the composition is selected from the group consisting of: a deubiquitinating enzyme or a derivative thereof, and/or a deubiquitinating enzyme agonist.
10. An isolated complex characterized in that the complex is a complex formed by the combination of a deubiquitinating enzyme and RORyt.
11. Use of a protein complex according to claim 7 for screening a drug or compound which promotes or inhibits deubiquitinating enzyme and RORyt to form said complex.
12. Use of a deubiquitinating enzyme antagonist for the preparation of a therapeutic systemic red A drug for lupus, rheumatic heart disease, or psoriasis.
13. The use according to claim 12, wherein the deubiquitinating enzyme antagonist is: 4,4", 5', 6'-tetrahydroxy[1,1':4',1"-triplet Benzene-2',3'-diphenyl phenylacetic acid (Vialinin A) or a derivative thereof.
14. Use of a composition comprising one or both components selected from the group consisting of IL-6 and TGFβ in the preparation of a reagent that facilitates the entry of deubiquitinating enzyme into the nucleus.
15. 4,4",5',6'-tetrahydroxy[1,1':4',1"-terphenyl]-2',3'-diphenyl phenylacetic acid, derivatives thereof, or pharmaceutically acceptable thereof Use of the accepted salt in the manufacture of a medicament for the treatment of an autoimmune disease, tumor, or inflammatory response.

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