砷新靶蛋白核仁素SUMO化修饰的发现及其作用机制的研究

Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications(PTMs), including phosphorylation, methylation and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of lately verified forms of PTMs, and exerts various effects on the target proteins. Our preliminary studies clearly indicated that nucleolin was subjected to SUMOylational modification. More importantly, we found arsenite treatment for 24 h could obviously increase nucleolin SUMOylation. This entrigerred us to explore the promising nucleolin SUMOylation site by various molecular methods, including site-directed point mutagenesis, Western Blotting assay, and Ni2+-beads pulldown. Furthermore, we will check whether nucleolin-SUMO would affect arsenite-induced cell apoptosis and cell transfomration using both in vitro and in vivo methods. In addition, we aim to investigate the underlying molecular mechasims for the role of nucleollin-SUMO in arsenite-associated cellular functions. Collectively, our findings here not only discovered a new modification of nucleolin protein and its novel functional paradigm in mRNA biogenesis processes, but also expanded our understanding regarding the dichotomous roles of arsenite in term of cancer development dependent on multiple intracellular conditions, such as oxidative stress vs. pro-proliferative signals, which made appropriate regulations of protein modifications, including SUMOylation.

砷化物是一类致癌但同时又可治癌的特殊化合物，但目前尚不能完全解释砷的这种双重生物学功能，故发现砷化物新靶点并阐述其作用机制是当前的研究热点。核仁素是一多功能核仁蛋白，具有传统原癌基因促增殖抑凋亡的作用。但我们研究发现，核仁素对砷暴露所致细胞凋亡有显著促进作用；核仁素能被SUMO修饰，且砷化物可促进该修饰作用。因此，我们推测核仁素及其SUMO修饰可能是介导砷化物"双刃剑"生物学功能的主要枢纽分子。本课题在进一步明确核仁素SUMO修饰位点的基础上，拟深入探讨砷暴露下核仁素及其SUMO修饰的功能及其分子机制。此项研究不仅有助于进一步了解砷化物的致癌机理、指导砷化合物在临床上安全有效的使用,而且将为合成和寻找高效且无或少副作用的砷衍生物提供理论依据和实验基础。

核仁素是一种普遍存在的蛋白质，参与许多重要的生物过程，如细胞周期调控和核糖体生物合成。核仁素的活性受细胞内定位和翻译后修饰的调节，包括磷酸化，甲基化和ADP-核糖基化。SUMO是近年来新发现的一类蛋白翻译后修饰，具有调节蛋白空间构象、定位、蛋白间结合等功能。本课题在进一步明确核仁素 SUMO 修饰位点的基础上，拟深入探讨砷暴露下核仁素及其 SUMO 修饰的功能及其分子机制。我们发现在Lys-294位点上人核仁蛋白发生SUMO化修饰，通过维持其核定位促进了核仁蛋白的mRNA结合特性，并阐明核仁素的SUMO化修饰通过增强核仁素与gadd 45αmRNA的结合，进而提高gadd 45αmRNA的稳定性，从而在砷诱导细胞凋亡过程中发挥重要作用。另一方面，Mn-SOD的异位表达减弱了亚砷酸盐产生的超氧化物自由基水平，消除了核仁蛋白-SUMO，进而通过减少GADD45表达抑制了亚砷酸盐诱导的细胞凋亡。总而言之，我们的结果首次发现在K294R的核仁素SUMO化修饰在其细胞核隔离和gadd45 mRNA结合活性中起关键作用。因此，我们的研究结果不仅揭示了核仁蛋白的新修饰及其在mRNA代谢中的新功能，而且扩展了我们对核仁蛋白在癌症发展方面的理解。