NIRF泛素化p53的作用过程中NIRF与p53结合域的测定

Identification of a NIRF-interacting Domain of p53 for its NIRF-mediated Ubiquitination

NIRF(Np95/ICBP90-like RING finger protein)是2002年发现的一种核蛋白,其功能涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制等领域.已有研究报道,NIRF能与p53相互作用,NIRF本身也是一个高度调节蛋白,在细胞正常的生理状态下发挥泛素化E3连接酶的作用,结合p53并将其降解,但NIRF与p53结合的蛋白结合域目前尚不清楚.本文研究证明,NIRF能与p53结合成复合体参与泛素化蛋白降解途径,并测定出NIRF与p53结合的区域.为了检测NIRF的蛋白结合域,将空载体和NIRF缺失突变体质粒分别转染于HEK293细胞,蛋白表达水平通过Western印迹用两种抗体分别检测.结果显示,所有的突变体都能在细胞中表达,并且两种抗体检测结果完全一致.同时,免疫共沉淀技术用于进一步分析实验结果.由于泛素化蛋白通常伴随蛋白酶体通路介导的降解,免疫共沉淀的蛋白纯化过程中用蛋白酶体抑制剂MG-132以抑制蛋白降解.本研究结果显示,NIRF通过PHD区域与p53形成复合体.该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动,从而形成与细胞增殖相关的新的信号通路,在肿瘤的发生发展中可能发挥某种程度的作用.

NIRF（Np95/ICBP90-like RING finger protein） is a nuclear protein identified in 2002 that is involved in the regulation of cellular proliferation,poly-ubiquitinated protein degradation,and the control of cellular carcinogenesis under certain conditions.NIRF was known to interact with p53,and thought to be a highly-regulated E3 ligase for the normal physiological degradation of p53.However,the NIRF-interacting domain within p53 remained unclear.In order to identify the NIRF-binding domain,NIRF deletion mutants were transiently transfected into HEK293 cells,co-immunoprecipitation was performed with the presence of proteasome inhibitor MG-132 to prevent the degradation of ubiquitinated proteins during the isolation.The levels of deletion mutant proteins were assayed by Western blotting and identified by two differed antibodies.We found that NIRF bound to p53 through its PHD domain.The formed complexes might be important for protein sorting and degradation,then perhaps also involved in DNA repair,apoptosis,or signal pathways for cell proliferation,which related to the development and progression of cancers.