SND1蛋白与HuR蛋白的相互结合作用

人源SND1（staphylococcal nuclease domain containing 1）蛋白由N端的SN（staphylococcal nucleases）结构域和C端的TSN (Tudor SN5) 结构域组成，其中SN结构域又包含SN1～SN4四个重复的功能片段. 本课题组前期研究结果表明，SND1蛋白可以通过SN结构域与G3BP（Ras GAP SH3 domain binding protein）蛋白相互结合，共同参与细胞应激颗粒（stress granules，SGs）的形成. SGs是真核细胞在受到氧化应激、病毒感染等外界刺激时在胞浆内形成的与RNA代谢相关的颗粒状结构. 对于SGs的成分鉴定及相互作用的分析一直是学者们研究的热点. 本研究中，免疫共沉淀实验结果表明，以抗SND1抗体可以共沉淀出HeLa细胞内另一个重要的应激相关人类抗原R（human antigen R，HuR）蛋白. 另外，利用脂质体转染法将pcDNA3 FLAG HuR重组质粒瞬时转染入HeLa细胞，成功过表达外源性的FLAG HuR融合蛋白，再以抗FLAG标签抗体又可以反向共沉淀出内源性SND1蛋白，证明SND1与HuR之间存在蛋白质间的相互结合作用. 细胞免疫荧光实验结果表明，当给予HeLa细胞05 mmol/L亚砷酸钠氧化应激时，SND1与HuR蛋白共同定位于胞浆中的SGs结构中. GST pulldown实验结果进一步表明截短的SN结构域可以结合HuR蛋白，其中以SN1功能片段的结合能力最强，表明SND1蛋白是通过SN结构域与HuR蛋白形成应激复合物，参与SGs的胞浆组装.并不定位于SGs的TSN结构域亦可结合HuR蛋白，提示SND1 HuR的蛋白相互作用可能并不局限于SGs，具有其它方面的功能意义.

The Interaction between SND1 and HuR Protein

Human staphylococcal nuclease domain containing 1(SND1) protein contains the N-terminal SN (1-4) domains and C-terminal TSN (Tudor-SN5) domain. Our previous study indicated that the SND1 protein interacts and co-localizes with G3BP (Ras-GAP SH3 domain-binding protein) protein into stress granules (SGs) structure, via its SN domain. Stress granules are a type of cytoplasmic RNA foci that aggregate in response to environmental stress stimuli, such as oxidative stress, or viral infection. Here, the result of co immunoprecipitation experiment indicated that anti-SND1 antibody was able to efficiently precipitate endogenous HuR (human antigen R), another stress associated protein. In reciprocal experiment, after HeLa cells were transfected with pcDNA3 FLAG HuR recombinant plasmid, SND1 protein was also detected in the anti-FLAG immunoprecipitations from total cell lysates. These data demonstrated that SND1 binds HuR protein in vivo. The result of immunofluorescence (IF) assay further indicated that SND1 co-localizes with HuR protein and is recruited into the same SGs in response to the 0.5 mmol/L arsenite sodium induced oxidative stress. To delineate the interaction domains of SND1 with HuR protein, GST-pulldown experiments were then performed. The results showed that total either SN domain or individual SN1 fragment physically binds HuR protein with high affinity, while other SN2, SN3, SN4 fragments show diminished different affinity associations, suggesting that SN domain contributes to the formation of SND1/HuR protein complex in SGs structure. In addition, TSN domain alone, which does not function in SGs, mediates the interaction relationship between SND1 and HuR as well, hints the existence of other potential function.