Application of ubiquitination pathway related factor in regulating and controlling functions of regulatory T cell

A regulatory and deubiquitinating enzyme technology, applied in the field of molecular biology and biomedicine, can solve the problems of unclear research on inducible regulatory T cells

Inactive Publication Date: 2018-06-12
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of regulation of inducible regulatory T cells (iTreg) under inflammatory conditions is still unclear

Method used

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  • Application of ubiquitination pathway related factor in regulating and controlling functions of regulatory T cell
  • Application of ubiquitination pathway related factor in regulating and controlling functions of regulatory T cell
  • Application of ubiquitination pathway related factor in regulating and controlling functions of regulatory T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0256] Example 1, USP44 is specifically highly expressed in iTreg

[0257] The present inventors first performed flow cytometric antibody staining on peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood (healthy people unless otherwise specified), and then used flow cytometry (FACS) to obtain Naive T cells were isolated from PBMC ( T), effector T cells (Teff) and nTreg, induced in vitro T to make it differentiate into iTreg, lyse the cells and extract RNA for real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qRT-PCR) to detect the expression of related genes, the results showed that USP44 was specifically highly expressed in iTreg ( figure 1 A). Different T helper cell (Th) subsets (Th1, Th2, Th17) and iTreg were induced and differentiated in vitro, and qRT-PCR was also performed, which also proved that USP44 was specifically highly expressed in iTreg ( figure 1 B). It shows that the expression of USP44 and FOXP3 in iTreg is ...

Embodiment 2

[0258] Example 2, USP44 interacts with FOXP3

[0259] Since USP44 and FOXP3 are synergistically up-regulated in iTreg, the inventors hypothesized that the two may have some kind of interaction. In order to verify whether USP44 acts on FOXP3 and is related to its function, the inventor designed an experiment to verify the interaction between the two. First, FLAG-USP44 and MYC-FOXP3 were expressed in HEK293T cells, and the two-way co-immunoprecipitation and protein immunoblotting experiments were performed, and the results showed that the two could interact in the transfection system ( figure 2 A). Then the Jurkat T cells stably expressing HA-FOXP3 were collected, and two-way co-immunoprecipitation and protein immunoblotting were performed with the corresponding antibody, and the results showed that the two could also interact in the stable cell line ( figure 2B). Then isolated from human peripheral blood PBMC T cells were induced to differentiate into iTreg in vitro, co...

Embodiment 3

[0260] Example 3, USP44 stabilizes FOXP3 protein

[0261] In order to understand the effect of USP44 on the protein of FOXP3, the inventors first co-expressed MYC-FOXP3 and FLAG-USP44 in an overexpression system (the dose was gradually increased by 0.5, 1, 1.5 μg), and performed western blotting with the corresponding anti-tag antibody Experiments, the results show that USP44 can stabilize FOXP3 protein, and has a dose-dependent ( image 3 A). Since USP44 has deubiquitinase activity, in order to verify whether its stability to FOXP3 protein is related to its deubiquitinase activity, the inventors constructed a USP44 enzyme inactivation mutant (C282S) and designed a protein synthesis inhibitor Cycloheximide CHX treatment (0, 4, 8, 12h) experiments, the results showed that co-expression of wild-type (WT) USP44 can significantly prolong the half-life of FOXP3 protein, while the enzyme inactivation mutation C282S significantly reduced this effect, which shows that USP44 The ef...

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Abstract

The invention relates to an application of a ubiquitination pathway related factor in regulating and controlling functions of a regulatory T cell. Specifically, the invention relates an application ofthe ubiquitination pathway related factor and an agonist or an antagonist thereof in preparing a preparation or a reagent for regulating FOXP3 or the regulatory T cell, wherein the ubiquitination related factor is selected from gene coding proteins USP44 and USP7 of a ubiquitination protein family. The novel regulatory factor provided by the invention, by regulating transcriptional regulatory activity of the FOXP3 on a target gene thereof, can regulate and control the regulatory T cell and the balance of an immune system.

Description

technical field [0001] The invention relates to the fields of molecular biology and biomedicine. More specifically, the present invention relates to the application of deubiquitination pathway-related factors in regulating the function of regulatory T cells. Background technique [0002] Regulatory T cells (Tregs) play an important role in maintaining the body's immune tolerance and immune homeostasis, preventing the occurrence of autoimmune diseases, and playing an important role in anti-graft rejection and tumor immunity. Human forkhead box P3 (Foxp3) is an important transcription factor for Treg development and functional maintenance, and plays an important role in regulating the development and function of Treg. In humans, the loss of FOXP3 function can cause immune dysfunction, causing a variety of endocrine disorders, intestinal diseases and X-linked syndrome IPEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy, X-linked). In mice, loss of function of FOXP3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K45/00A61P37/04A61P35/00A61P29/00C12N9/48
CPCA61K38/4813A61K45/00C12N9/485C12Y304/19012
Inventor 李斌杨静徐鹏李丹梁瑞
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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