PTD-mediated intracellular delivery of mutant NFAT minimum DNA binding domain inhibited the proliferation of T cells.
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-ΔNFATminiDBD), 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-ΔNFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-ΔNFATminiDBD 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-ΔNFATminiDBD 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.
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Xia
X
Liu
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; The Center for Disease Control and Prevention of Jinhu City, Huaian 211600, Jiangsu, PR China.
Qianqian
Q
Zhao
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; The Hospital of Ningbo University of Technology, Ningbo 315211, Zhejiang, PR China.
Xin
X
Peng
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China. Electronic address: xiasheng1519@163.com.
Sheng
S
Xia
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Weihong
W
Shen
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Yangyong
Y
Zong
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Jing
J
Cheng
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Weijiang
W
Wu
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Miaomiao
M
Zhang
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Fengyi
F
Du
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Wenrong
W
Xu
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China.
Hui
H
Qian
School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China; Zhenjiang Key Institute of Clinical Laboratory Medicine, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang 212013, Jiangsu, PR China. Electronic address: shao_qx@ujs.edu.cn.
Qixiang
Q
Shao
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage*; D007376:Interleukin-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D050778:NFATC Transcription Factors / Q000008:administration & dosage* / Q000037:antagonists & inhibitors* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage* / Q000235:genetics
D057846:Cell-Penetrating Peptides; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D004247:DNA; D007770:L-Lactate Dehydrogenase
IL-2; Immunosuppressant; Mutant minimum DNA binding domain of NFAT1; NFAT; PTD