BAG6复合体在DNA损伤反应过程中的功能研究

DNA damage response (DDR) is a crucial regulatory mechanism that coordinates the cell cycle check points, repair and cellular apoptosis for the maintenance of genomic integrity. Deregulation of DDR is associated with many genetic diseases including cancer and neurological disorders. Central to the DDR are checkpoint kinases ATM and ATR that phosphorylate a plethora of substrates. Through genome-wide screens we and others have identified more than 700 potential ATM/ATR substrates, which greatly expanded the DDR landscape and revealed the involvement many previously unknown pathways. One of the most intriguing observations is the over-representation of proteins involved in ubiquitin proteasome system (UPS) pathways. .Large proline-rich protein BAG6 is a novel ATM/ATR substrate candidate identified in our screen. Biochemistry analysis indicates that BAG6 functions as a negative regulator of Hsp70 chaperone activity and facilitates p53 acetylation upon DNA damage. Most recently, BAG6, a nuclear protein, has been shown to form a stable complex with GET4 and UBL4A and plays an important role in cellular protein quality control by targeting multiple substrates. We have carried out preliminary studies to investigate the role of BAG6 complex in DDR. Our data showed that BAG6 is phosphorylated in an ATM-dependent manner and loss of BAG6 complex result in defective G2/M checkpoint. Importantly, GET4 and UBL4A, which normally reside in cytoplasm, translocate into the nucleus. Knocking down BAG6 complex by siRNA led to a defect in DNA damage-induced BRCA1 foci formation, suggesting the involvement of BAG6 complex in DDR. .The goal of this proposal is to establish the role BAG6 complex as a co-chaperone in DDR and identify direct targets of BAG6 complex in this process. We hypothesize that upon DNA damage, the BAG6 complex is enriched in nucleus through protein translocation and acts as a surveillance machinery for protein quality control. This surveillance is necessary as numerous proteins are relocated to sites or vicinity of DNA damage. To achieve this goal, mass spectrometry is used to identify direct targets of BAG6 complex. GST fusions of UBL domains of BAG6 and UBL4A and BAG domain are used for pull-down experiments. Proteins identified from these pull-downs and BAG6-associated complex are prime candidates. Since loss of BAG6 led to protein level increase of its substrates, genome-wide protein profiling will be performed in BAG6 knockdown cells using quantitative mass spectrometry. The identified substrates are validated by in vitro chaperone activity assay and by their in vivo functions in DDR. If proven, our studies would uncover a novel surveillance mechanism in DDR and establish BAG6 complex with 2 UBLs and a BAG domain as the executioner for this function, linking protein quality control, UPS to DDR.

DNA损伤反应是哺乳动物细胞维持基因组的稳定性，保持细胞存活和抑制肿瘤的关键通路，其发生发展涉及到大规模蛋白转运、细胞内重定位、DNA反应/修复复合物重构及蛋白重折叠，过程极为复杂，其机制仍不明了。BAG6蛋白既具有与泛素系统相互作用的UBL结构域又具有分子伴侣系统BAG结构域，因而具有协助蛋白转运、折叠及对此过程监控的能力。我们前期研究发现BAG6与GET4和UBL4A所形成的BAG6复合体可调控DNA损伤调节因子BRCA1向DNA损伤位点的募集，提示其在DNA损伤修复中的重要作用。本课题拟在此基础上进一步研究其在DNA损伤反应中的功能，利用蛋白质组学定量平台，筛选其在该过程中的底物靶点蛋白。获得一批相关调控分子，形成由BAG6复合体发散开的DNA损伤反应调控网络。为全面了解DNA损伤反应体系中的精确控制、各层面蛋白分子调控机制及其与分子伴侣系统、泛素化系统的关联提供全新的理论依据。

Bcl2相关的永生基因（BAG）家族蛋白是含有BAG结构域的蛋白质，并在真核生物中进化保守。BAG家族蛋白在多种细胞过程中起关键作用，例如细胞存活，增殖，迁移，凋亡，应激反应和神经分化。BAG结构域为BAG家族蛋白的基本功能结构域，我们通过对BAG家族蛋白成员及BAG结构域间的相互作用蛋白进行分析，以探明不同BAG结构域相互作用蛋白的异同点，进而研究BAG家族蛋白多样性生物功能的分子机制。我们构建了p-GEX-4T2-BDs重组子并转化E.coli BL21（DE3）经IPTG诱导表达GST-BDs融合蛋白并纯化。采用GST pulldown技术联合高效液相色谱串联质谱(LC-MS/MS)的策略对BAG结构域相互作用蛋白进行定性定量分析。我们发现BAG家族蛋白不同成员的BAG结构域所介导的蛋白-蛋白相互作用既有共性又有特异性，BAG家族蛋白通过BAG结构域介导多种蛋白相互作用并参与细胞内多条重要的信号通路来调控细胞内蛋白质稳态、糖代谢、免疫反应、应激反应、细胞周期等过程。我们的研究成果为理解BAG结构域在介导BAG蛋白质的不同细胞功能中的作用提供了借鉴与参考。