阅读说明：本技术 T细胞特异接头蛋白slp-76的类泛素化修饰位点的鉴定及应用 (Identification and application of ubiquitin-like modification site of T cell specific adaptor protein SLP-76 ) 是由 刘合宾 熊伊韦 于 2019-09-05 设计创作，主要内容包括：本发明属于生物医药技术领域,本发明首次鉴定出特异接头蛋白SLP-76的类泛素化修饰的位点在第266位和第284位的赖氨酸上,并且突变这两个类泛素化修饰位点可直接改变T细胞功能,该方法具有可靠性、简便性和经济性,以及较高的应用价值。(The invention belongs to the technical field of biological medicines, and identifies that the ubiquitin-like modified sites of specific joint protein SLP-76 are positioned on the 266 th lysine and the 284 th lysine for the first time, and the mutation of the two ubiquitin-like modified sites can directly change the function of a T cell.)

A T cell specific adaptor protein SLP-76 ubiquitin-like modification site, wherein the modification site is the 266 th lysine.

A T-cell specific adaptor protein SLP-76 ubiquitin-like modification site, wherein the modification site is the 284 th lysine.

A T-cell specific adaptor protein SLP-76 ubiquitin-like modification site, wherein the modification site is lysine at 266 th site and 284 th site.

4. The ubiquitin-like modified site of mutant SLP-76 is used for regulating the IL-2 production of T cells.

5. The use according to claim 4, wherein the lysine position at position 266 of SLP-76 is mutated.

6. The use according to claim 4, wherein the lysine site at position 284 of SLP-76 is mutated.

7. The use of claim 4, wherein lysine positions 266 and 284 of SLP-76 are mutated.

8. The use according to any one of claims 4-7, wherein the ubiquitination-like modification site of mutant SLP-76 eliminates the IL-2 production effect of SLP-76 and Ubc9 in cooperation with T cells.

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to identification and application of a ubiquitination-like modification site of T cell specific adaptor protein SLP-76.

Background

SLP-76 is a T cell specific immune linker protein structurally comprising multiple domains that mediate the interaction of SLP-76 with other protein signaling molecules, thereby performing a series of regulatory functions of T cell receptor signaling. The SLP-76 is a key node pivot molecule of a TCR signal transduction core signal axis (TCR-p56Lck-ZAP-70-LAT-GADs-SLP-76), not only can integrate cascade amplification of TCR near-end signals and far-end signals, but also plays an important regulation role in TCR signal transduction. SLP-76 mediated TCR proximal signaling events including tyrosine phosphorylation, interactions and activation of important signaling molecules; the remote signals include activation of important signal paths such as ERK and NF-B, cytoskeletal rearrangement, immunoadhesion, increased secretion of cytokine IL-2, and the like. These TCR signaling cascades ultimately lead to T cell differentiation, proliferation, activation, and acquisition of effector immune responses. Therefore, the change of SLP-76 protein structure or translation modification level can directly affect T cell function.

SUMOylation (SUMOylation) is an important post-translational modification process for achieving fine control of signal transduction in various cells, and is widely involved in regulating various links of protein functions and cell life activities, similarly to Ubiquitination (Ubiquitination). Their reaction mechanisms are similar, and the target of action of the substrate is lysine. The difference between them is that Ubiquitin-like process is modified by Ubiquitin molecules, and Ubiquitin-like is modified by SUMOs (small-like modifiers). In most cases, ubiquitination and sumoylation regulate the function of substrate proteins in an antagonistic manner. Moreover, ubiquitination and ubiquitination can exist simultaneously as a pair of mutually restricted regulation mechanisms in many cells, and can modify the same target protein or even competitively modify the same lysine residue on the same protein, such as the K21 site of IB, so that the balanced regulation of the signal conduction path mediated by the target protein in a positive-negative way is realized.

At present, the regulatory role of the ubiquitination pathway in T cell signaling and the mechanism thereof have been well studied: degradation caused by ubiquitination modification mediated mainly by ubiquitin ligases such as c-Cbl, Cbl-b and Itch. Target molecules of these ubiquitin ligases in T cells include such signal molecules as Lck, Fyn, ZAP-70 and SLP-76 which play an important role in T cell activation signaling. These molecules are modified by ubiquitination and are degraded, thereby down-regulating TCR signaling strength and T cell immune response levels. Therefore, the protein ubiquitination modification has important biological significance for regulating T cell functions, and provides potential drug action targets and treatment strategies for treating T cell related diseases.

Although the process of ubiquitin-like modification and ubiquitination have a plurality of points, how important target molecules in T cells are modified by ubiquitin-like modification is still little known so far, particularly the key linker protein SLP-76, no report about SLP-76 modified by ubiquitin-like modification exists at present, and a positive result cannot be obtained by identifying the ubiquitin-like modification of SLP-76 by a traditional method. In the previous research on the adaptor protein SLP-76, an analysis method capable of directly identifying the level of SLP-76 ubiquitin-like was lacked, and the technical analysis probably caused that: firstly, the ubiquitination is a reversible process, besides, the ubiquitination molecules can be quickly removed by the hydrolysis reaction catalyzed and mediated by the enzyme senp1, the abundance of the intracellular protein ubiquitination modification is generally low, and the requirement on the sensitivity of a detection method is high; secondly, SLP-76 itself has intramolecular effect to control the level of affecting ubiquitination.

Disclosure of Invention

The invention aims to solve the problem of providing identification and application of a T cell specific adapter protein SLP-76 ubiquitin-like modification site on the basis of the prior art.

The first aspect of the invention provides a T cell specific joint protein SLP-76 ubiquitin-like modification site, which is the 266 th lysine.

The ubiquitin-like modification site of the T cell specific adapter protein SLP-76 is lysine at position 284.

T cell specific joint protein SLP-76 ubiquitin-like modified sites, 266 th and 284 th lysine.

In a second aspect, the invention provides a ubiquitination-like modification site of mutant SLP-76 to regulate T-cell IL-2 production.

In a preferred embodiment of the present invention, the lysine position at position 266 of SLP-76 is mutated.

In a preferred embodiment of the present invention, the 284 th lysine site of SLP-76 is mutated.

In the preferred technical scheme of the invention, the 266 th site and 284 th site of SLP-76 are mutated.

In the preferred technical scheme of the invention, the ubiquitin-like modification site of the mutant SLP-76 eliminates the IL-2 production effect of the SLP-76 and Ubc9 which are cooperated to promote T cells.

The sequence of SLP-76 protein is (NCBI Gene database, accession No. NM-005565.5).

According to the invention, by directly identifying the analysis method of the SLP-76 ubiquitination level, on the basis of the method, the ubiquitination modification sites of the specific adaptor protein SLP-76 are firstly identified to be on the 266 th lysine and the 284 th lysine, and the T cell function can be directly changed by mutating the two ubiquitination modification sites.

Drawings

FIG. 1 shows that the T cell specific adaptor protein SLP-76 ubiquitination detection method is used to increase the SLP-76 ubiquitination modification abundance.

FIG. 2 is a Coomassie brilliant blue staining chart after SLP-76 ubiquitination-modified protein enrichment and separation.

FIG. 3 is a graph showing the results of protein spectrum analysis of SLP-76 ubiquitination-modified protein having a high molecular weight (lysine modification site K266).

FIG. 4 is a graph showing the results of protein spectrum analysis of SLP-76 ubiquitination-modified protein having a high molecular weight (lysine modification site K284).

FIG. 5 shows that the SLP-76 ubiquitination-like sites K266 and K284 are verified by Western blotting, and that the SLP-76 ubiquitination-like site mutation causes a decrease in the level of ubiquitination-like modification.

FIG. 6 shows the effect of SLP-76 ubiquitination-like site mutation on the transcription level of IL-2 in T cells.

FIG. 7 shows the effect of SLP-76 ubiquitination-like site mutation on the production of T-cell IL-2 protein.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the methods used in the following examples are conventional unless otherwise specified, for example, see the specific procedures: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual,3 rd edition,2001, NY, Cold spring harbor).

The primers and sequences used were synthesized by Shanghai Biotech, Inc. Expression vector pcDNA 3.1 was purchased from Invitrogen, plasmids pSRa-UBC9, pSRa-SUMO1 and pSRa-SLP-76, and Jurkat T cell, HEK293T cell, which are explicitly mentioned and are named in agreement in The text "The Immune Adaptor SLP-76 binders to SUMO-RANGAP1 at Nuclear Pore Complex files to Regulation Nuclear expression of transcriptional microorganisms in T Cells" published in Molecular Cells.