PSPC1与ATRIP相互作用分子机制及其功能研究

The DNA damage response(DDR) is an important cellular mechansim for maintaining genomic integrity in the face of genotoxic stress, such as ionizing radiation induced DNA damage.It is a complex network of signal transduction pathways involving a vast array of signal molecules , and new regulatory molecules are constantly discovered. Previously,We have found that expression of PSPC1 protein in the cell was increased during DNA damage, and the changed expression influenced the degree of DNA damage,implying that PSPC1 might be involved in DDR. Furthermore, it was shown that PSPC1 can regulate the cell cycle in DDR .However, the exact molecular mechanisms are still unclear. In the case of DNA damage, PSPC1 can have an interaction with ATR-interaction protein (ATRIP), a key molecule in DDR. ATRIP is responsible for the initiation of DDR and it also has a crucial role in the regulation of cell cycle. We hypothesize that PSPC1 may regulate the cell cycle by its interaction with ATRIP. In this study,firstly,we will verify the interaction between PSPC1 and ATRIP in vivo and vitro.Second, we will identify the interaction domain between these two proteins. Finally, we will confirm the function of this interaction on cell cycle regulation and the whole process of DDR. It may provide benificial information for better understanding of the function and molecular mechanism of PSPC1 in DDR.

DNA损伤应激反应（DDR）是机体应对电离辐射等所致DNA损伤的重要机制，多种信号分子参与其中，且不断有新的调控分子被发现。我们前期研究发现，核旁斑点蛋白1（PSPC1）在DNA损伤情况下表达显著升高，且其表达影响DNA损伤程度，提示PSPC1极可能参与细胞的DDR。进一步发现，PSPC1可通过调控细胞周期参与DDR，但具体机制尚不明确。细胞DNA损伤后，PSPC1可与DDR核心分子ATR相互作用蛋白 （ATRIP）结合。ATRIP是DDR的启动分子，且对细胞周期具有重要调控作用。我们认为PSPC1很可能是通过与ATRIP相互作用，调控细胞周期。本研究拟围绕这一假设，首先通过体内与体外实验，确认PSPC1与ATRIP在DDR中存在相互作用；其次鉴定两者相互作用的结构域；最后明确这种相互作用对细胞周期及整个DDR过程的影响。通过以上研究，将有助于阐明PSPC1在DDR中功能及作用的分子机制。

DNA损伤应激反应（DDR）是机体应对电离辐射等所致DNA损伤的重要机制。我们前期研究发现,PSPC1可通过调控细胞周期参与DDR，但具体机制尚不明确。质谱结果提示PSPC1可与细胞周期调控核心分子ATR相互作用蛋白 （ATRIP）结合。本研究首先构建了PSPC1-pcDNA3.1-3×Flag和ATRIP -pcDNA6-myc表达载体，共转染到Hela细胞，采用免疫共沉淀技术（co-IP）检测PSPC1与ATRIP的结合情况。然而，可能由于前期的质谱结果存在假阳性，co-IP的结果并未能证实PSPC1与ATRIP结合。 为了进一步寻找在DDR中发挥重要作用的PSPC1直接互作蛋白，我们采用顺铂处理HeLa细胞诱导细胞DDR，获取干预组和对照组中与PSPC1存在直接相互作用的蛋白质，采用质谱技术对其进行鉴定，并进行生物信息学分析。质谱结果筛选到11个与PSPC1直接互作候选蛋白，包括已经报道的NONO和SFPQ和尚未见报道白介素增强子结合因子(Interleukin Enhancer Binding Factor 2,ILF2) 核不均一核糖核蛋白K (Heterogeneous Nuclear Ribonucleoprotein K, hnRNPK)，富含丝氨酸/精氨酸剪接因子1（Serine And Arginine Rich Splicing Factor 1，SRSF1）等。采用co-IP技术验证了SRSF1与PSPC1之间存在直接互作用。