PTD-mediated intracellular delivery of mutant NFAT minimum DNA binding domain inhibited the proliferation of T cells

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• 作者：Xia Liu^a ; ^b ; ¹ ; Qianqian Zhao^a ; ^c ; ¹ ; Xin Peng^a ; ^d ; ¹ ; Sheng Xia^a ; ^b ; ^{xiasheng1519@163.com" class="auth_mail} ; Weihong Shen^a ; Yangyong Zong^a ; Jing Cheng^a ; Weijiang Wu^a ; Miaomiao Zhang^a ; ^b ; Fengyi Du^a ; Wenrong Xu^a ; ^b ; Hui Qian^a ; ^b ; Qixiang Shao^a ; ^b ; ^{shao_qx@ujs.edu.cn" class="auth_mail}
• 关键词：DBDs ; DNA binding domains ; 螖NFATminiDBD ; a minimum DNA binding domain with point mutation in R466A and T533G of human NFAT1 ; eGFP ; enhanced green fluorescent protein ; NFATminiDBD ; a wild type mini DNA binding domain of human NFAT1 ; NFAT ; nuclear factor of activated T cell ; NF-魏B ; nuclear factor 魏-light-chain-enhancer of activated B cells ; PBMCs ; peripheral blood mononuclear cells ; PHA ; phytohemagglutinin ; PMA ; phorbol 12-myristate 13-acetate ; PI ; propidium iodide ; PTD ; protein transduction do
• 刊名：International Immunopharmacology
• 出版年：March, 2014
• 年：2014
• 卷：19
• 期：1
• 页码：110-118
• 全文大小：995 K

文摘

The nuclear factor of activated T cell (NFAT) family of calcium-regulated transcription factors plays a key role in the development and function of the immune system. Calcineurin, a protein phosphatase, activates NFAT by dephosphorylation. The activated NFAT is translocated into the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) and other target genes. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 are effective immunosuppressant drugs and dramatically increase the success rate of organ transplantation procedures. However, since calcineurin is expressed in most tissues in the body and calcineurin inhibition alters many cellular processes besides immune cell activation, the therapeutic use of calcineurin inhibitors is limited by serious side effects. Thus inhibiting NFAT by other mechanisms such as blocking its binding to DNA could be a more selective and safer approach to target NFAT for therapeutic applications. In peripheral T cells, productive immune responses are dependent upon the cooperative binding of the NFAT/AP-1 transcriptional complex to the promoter regions of genes such as interleukin-2 (IL-2), while NFAT in the absence of AP-1 leads to T cell anergy. Protein transduction domains (PTDs) are able to penetrate cell membranes and can be used to transport exogenous proteins across the cell and nuclear membranes. In this study, we constructed a fusion protein of PTD and a minimum DNA binding domain of human NFAT1 (PTD-螖NFATminiDBD), which contains two mutations (R466A and T533G) in the AP-1 binding sites. The delivery and functions of this fusion protein in T cells were investigated. The results indicated that PTD-螖NFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-螖NFATminiDBD attenuated IL-2 production in T cells and then inhibited T cell proliferation, likely through competing against endogenous NFAT for binding to the IL-2 gene promoter. These results demonstrated that PTD-螖NFATminiDBD was an effective NFAT inhibitor with a novel mechanism of action and might potentially be used as an immunosuppressant for organ transplantation with higher safety and better tolerance than calcineurin inhibitors.