中国科学院生物物理研究所,中国科学院大学,中国科学院生物物理研究所
国家重点基础研究发展计划(973)资助项目(2012CB910204)
Institute of Biophysics,Chinese Academy of Sciences, Graduate University of Chinese Academy of Sciences,Institute of Biophysics,Chinese Academy of Sciences
This work was supported by a grant from National Basic Research Program of China (2012CB910204)
人源N-Myc结合蛋白(N-Myc interactor, Nmi)能在干扰素(interferon,IFN)诱导后高表达,并参与下游的JAK-STAT通路,且有抑制癌细胞增殖的功能.Nmi能够结合人干扰素结合蛋白35(interferon-induced protein 35,IFP35)并保护IFP35不被降解.本研究构建了Nmi和IFP35的全长及N端截短体克隆,通过两种蛋白的共表达和GST pull down实验确定二者能够相互作用的区域,并进一步大量共表达和纯化了重组蛋白复合物.结合实验表明,与先前报道的两者通过C端串联的NID结构域相结合不同,两者的N端结构域都足以介导复合物形成.免疫荧光观察未经干扰素刺激的RAW264.7细胞,发现Nmi与IFP35共定位于细胞核内.免疫荧光观察Nmi在293A细胞中的分布特点,发现正常细胞中Nmi分布于细胞核内.如先前研究报道,干扰素诱导24 h后,Nmi在胞质内形成颗粒状聚集,此时加入低浓度过氧化氢(H2O2)会使Nmi的亚细胞定位回到细胞核内,表明Nmi的亚细胞定位受到细胞氧化环境的影响.
Human N-Myc Interactor (Nmi), an interferon (IFN)-induced protein, participates in downstream JAK-STAT pathway and inhibits proliferation of cancer cells.Nmi can bind Interferon-Induced Protein 35 (IFP35) and protect the latter from degradation.Previous research showed that these two proteins associated through their C-terminal domains.In this study, we constructed full length clones and N-terminal truncates of both Nmi and IFP35.Co-expression and GST pull down experiments showed that N-terminal domains of Nmi and IFP 35 are respectively sufficient for their association.We overexpressed and purified recombinant protein complexes afterward.Through immune fluorescence, we found that Nmi and IFP35 co-localized in nucleus in RAW264.7 cells.We also noticed that Nmi localized in nucleus and enriched on surface in normal 293A cells.Although Nmi granularly aggregated in cytoplasm after 24 h induction by interferon as reported, it came back to nucleolus by adding low concentration hydrogen peroxide (H2O2).That result indicated that subcellular localization of Nmi can be affected by the oxidation environment of cells.
沈涓,刘迎芳.干扰素诱导蛋白Nmi与IFP35的结合区域及Nmi亚细胞定位[J].生物化学与生物物理进展,2014,41(7):659-665
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