TAT介导的活化T细胞核因子衍生多肽对钙调神经磷酸酶的影响

Effect of TAT-Mediated Nuclear Factor of Activated T Cells-Derived on Calcineurin

目的:研究钙调神经磷酸酶(CaN)与活化T细胞核因子(NFAT)相互作用保守位点衍生的多肽和人免疫缺陷病毒(HIV)TAT的融合多肽对CaN-NFAT信号途径的影响。方法:根据CaN-NFAT的结合位点经计算机优化后得到PVIGIT,并和TAT融合成新的多肽PepNFAT-TAT,用异硫氰酸荧光素(FITC)荧光标记后加至携带CaN催化亚基cDNA的重组质粒pRED-CnA转染过的神经细胞LN-229中,再用离子霉素刺激,用激光共聚焦显微镜观察该融合多肽的细胞定位,并检测该多肽对CaN酶活性的影响,报告基因pNFAT-luc实验检测其对NTAF激活的影响,实时定量RT-PCR检测其对NFAT下游基因白细胞介素-4(IL-4)表达的影响。结果:该融合蛋白定位于细胞质中,报告基因实验显示该多肽能够抑制对NFAT的激活,抑制离子霉素诱导IL-4的表达。结论:融合多肽PepNFAT-TAT能抑制CaN-NFAT信号途径,但对CaN的酶活性没有影响,是潜在的免疫抑制剂。

Objective： To study the effect of fused peptides of conservative peptide derived from CaN- NFAT interaction sites and TAT from HIV on CaN-NFAT signal pathway. Methods： PVIGIT were obtained from CaN-NFAT interaction using computer optimization, and fused with TAT and generated a novel peptide PepNFAT-TAT. The fused peptide was labeled by the fluorescent substance fluoreseein isothioeyanate（FITC） and added to the LN-229 nerve cells transfeeted by the recombinant plasmid pRED-CnA with the CaN catalytic subunit eDNA. The location of Pep-NFAT-TAT in LN-229 cells was observed using laser scanning confocal microscope（LSCM） after the stimulation of ionomycin, and CaN activity was measured. The effect of this peptide on NFAT was also detected by reporter pNFAT-luc assay and the expression of IL-4 located in the downstream of NFAT was measured by real-time quantitative RT-PCR. Results： The fused pep- tide was located in cytoplasm and had no effect on CaN activity. The peptide could inhibit NFAT activation using reporter assay and the expression of IL-4 in LN-229 induced with ionomycin. Conclusion： These results suggested that PepNFAT-TAT could inhibit CaN-NFAT signal path- way and have no influence on CaN activity, implying that it is a potential immunosuppressant.