Regulatory Mechanisms Of SMURF2 In The Early Signaling Of T Cell Receptor

T cells(especially CD8~+T cell)are the major contributors to exert killing effects in human immune system.Existing studies suggest that controlling the killing ability of T cells by regulating the activation of T lymphocytes has significant therapeutic value for a variety of diseases,particularly for tumors.The tumor microenvironment(TME)is the main battlefield where immune cells exert their tumor recognition and killing effects.The presence of tumor-specific T cells has been identified in many tumors,suggesting that CD8~+T cells are capable of antigen recognition but not effective in killing tumor cells,and therefore more new approaches need to be developed to enhance the function of these cells.T cell receptor(TCR)is an important structure on T cells to recognize antigens.T cell activation and effector functions depend on the activation of TCR signaling pathway.When sufficient number of TCR binds to antigen,the activated TCR can effectively cause T cell-specific killing effects and initiate the acquired immune response.However,since the intracellular segment of TCR is very short,the activation signaling cannot be efficiently transduced into the cell and must be delivered by coupling with CD3 immunoreceptor tyrosine-based activation motif(ITAM)by non-covalent bond.Previous studies have revealed that post-translational modification plays an important role in the regulation of TCR proximal signaling.The post-translational modifications of TCR related signaling proteins contain numerous forms,including phosphorylation,ubiquitination and palmitoylation.Lymphocyte-specific protein tyrosine kinase(LCK)andζ-associated protein 70(ZAP-70)are two important protein kinases in TCR proximal signaling that mediate the phosphorylation of substrate proteins.Phosphorylation is an important form of modification in TCR signaling,as the initial TCR activation signaling is transmitted through phosphorylation of the intracellular segment of CD3 molecule.The phosphorylation level of LCK directly affects the activation of T cells.Inhibition of LCK activation using protein tyrosine kinase inhibitors significantly inhibits the phosphorylation of its substrate ZAP-70.Modulation of TCR proximal kinase phosphorylation by multiple modalities is an important means of regulating the immune response capacity of T cells.TCR proximal kinase,a key target for regulating early T cell signaling,is regulated by ubiquitination-mediated post translational modifications,including ubiquitinated degradation of targeted proteasomes and non-degradative polyubiquitination.In this study,we focused on the role of Smad ubiquitination regulatory factor 2(SMURF2),which has the HECT structural domain,in the early signaling of T cell receptor.SMURF2,an important E3 ubiquitin ligase,plays a key role in several aspects involved in cell growth,proliferation and senescence.Its family members play regulatory roles in various aspects of immune cell including their development,maturation and differentiation as well as the execution of effector functions.Based on these results,we explored the function of SMURF2 and the mechanism of its regulatory role in T lymphocyte.This study mainly includes three parts.Part Ⅰ:Effect of SMURF2 on the early signaling of TCRIn the first part of our study,we explored the effects of SMURF2 on signaling transduction of TCR early activation.Activation of TCR with antibodies in T cells significantly increased the phosphorylation of TCR proximal kinase ZAP-70.Subsequently,the SMURF2-knockdown T cells were constructed,and the knock-down cells were verified by western blot and real-time quantitative PCR.When SMURF2 was knocked down,the protein phosphorylation levels of LCK(Y394)and ZAP-70 were remarkably decreased.Activation of T-cell lines with knockdown SMURF2 expression using antibodies revealed that ZAP-70 was still phosphorylated,but at lower levels than control cells.In modeling the early phosphorylation of TCR using a time gradient,we found that the phosphorylation of both ZAP-70 and LCK were significantly down-regulated after knock-down of SMURF2.Dasatinib,a protein tyrosine kinase inhibitor,is mainly used in the treatment of chronic myeloid leukemia.We explored the inhibitory effect of Dasatinib on T cell activation and found that it significantly inhibited the phosphorylation of ZAP-70 at n M concentration,and the sensitivity of T cells to Dasatinib was significantly increased after SMURF2 knocked down.Part Ⅱ:Mechanism of SMURF2 in regulating TCR proximal kinaseIn the second part,we studied the regulatory mechanism of SMURF2 on early signaling transduction of TCR.As an E3 ubiquitin ligase,we first explored its potential substrates.The online prediction website was used to predict the substrate proteins of SMURF2,and the results showed that the protein tyrosine phosphatase SHP-1 may be the ubiquitination substrate of SMURF2.SHP-1 can dephosphorylate the intracellular proteins,and it includes two SH2 domains and a protein tyrosine phosphatase domain.Immunoprecipitation experiments and fluorescence resonance energy transfer experiment confirmed the existence of an interaction between SMURF2 and SHP-1.A truncated expression vector of SMURF2 was constructed and the HECT domain of SMURF2 was found to be responsible for the interaction with SHP-1.The ubiquitination-modified SHP-1showed reduced binding to LCK,which in turn prevented the dephosphorylation of LCK,causing an increase in the phosphorylation level of TCR proximal kinase.Further testing of the ubiquitination type of SHP-1 showed that K27 and K29 linkage was the main ubiquitination type of SHP-1.At the same time,the mutation of lysine at position 19 of SHP-1 could reduce the ubiquitination level of SHP-1,so this lysine might be the site where SMURF2 ubiquitinated SHP-1.Part Ⅲ:Establishment and identification of mouse model with t cell-specific Smurf2 gene modificationTo investigate in depth the role of the E3 ubiquitin ligase SMURF2 in T cell immune response as well as early TCR signaling,we constructed a T cell-specific Smurf2 gene-modified mouse model.Using Cre-loxp system,flox mice were obtained by introducing two Lox sites in the same direction on both sides of the Smurf2 exon 9 region and subsequently crossed with Cd4-Cre mice,and the offspring mice continued to be bred to silence T-cell Smurf2 expression.The results of nucleic acid electrophoresis demonstrated the successful construction of T-cell Smurf2-specific knockout mice.Subsequent researches contain multi-omics sequencing,T-cell permutation transfer,rheumatoid mouse models and combined immunotherapy based on the mouse to explore the role of SMURF2 on the immune response and their possible regulatory networks.In summary,the preliminary results indicated that E3 ubiquitin ligase SMURF2regulated the phosphorylation of LCK,a key kinase for early signal transduction of TCR,through the ubiquitination modification of SHP-1.This study has clarified the key role of SMURF2 in the activation of T cells,and provided a new target and idea for the regulation of T cell immune response and immunotherapy.