低氧预适应中Hsp90分子伴侣功能对相互作用底物蛋白的折叠、降解分选机制及作用

Hypoxia involves in various pathophysiological procedure of diseases,and contributes to the extreme environment induced stress response.Though the protection of hypoxic preconditioning has been confirmed,the mechanism of it remains unclear.Unfolded protein response(UPR),which tigtly related to protein quality control (PQC), plays an vital role in hypoxic preconditioning.As an important molecular in PQC,Hsp90 also contributes to preconditioning,and many Hsp90 client proteins are critical for hypoxic preconditioning formation.Our group has found,the intensity of stress infulenced Hsp90-client protein's interaction.In this project,we will establish hypoxic preconditioning cell line models for parallel comparision,combined stable isotope labeling by animo acid (SILAC) technique with immobilized Hsp90 antibody coimmunoprecipitation method to discover the dynamic changes of Hsp90 interactome.The Hsp90 SiRNA,ATPase mutant will be achieved by a lenti-virus system to analyse how Hsp90 affects client protens' folding/degradation triage and integrates the hypoxic prconditoning signaling pathways.We will focus on the change of Hsp90 level,post-translational staus,cochaperon bingding status,ATPase activity of Hsp90 to elucidate the role of Hsp90 in client protein quality control during hypoxic preconditioning,and lay a theoretical foundation for Hsp90 related prevention,therapy,drug development in hypoxic preconditioning and hypoxia related diseases.

低氧参与多种疾病的病理生理过程，且与极端环境诱发的应激反应密切相关。低氧预适应有明确的细胞保护作用，但其分子机制有待深入研究。未折叠蛋白质反应是低氧预适应形成的重要原因。分子伴侣Hsp90参与底物蛋白折叠与降解的分选，是蛋白质质控中的重要分子，与应激适应关系密切，且已知其多种底物蛋白参与低氧预适应调控。本研究在前期已确定Hsp90与底物蛋白结合、解离受应激强度影响的实验基础上，建立低氧预适应细胞模型，应用稳定同位素标记结合固化抗体免疫共沉淀技术，观测Hsp90与底物蛋白相互作用状态的时序性变化，以生物信息学手段分析Hsp90对低氧预适应信号通路的整合作用。以慢病毒转染技术建立Hsp90SiRNA干扰、ATPase突变体细胞株，分析Hsp90对底物蛋白折叠、降解分选机制。阐明Hsp90对低氧预适应信号通路的调控机制，为将与Hsp90相关的治疗、预防手段应用于低氧相关疾病奠定基础。

低氧参与多种疾病的病理生理过程，且与极端环境诱发的应激反应密切相关。低氧适应有明确的细胞保护作用，但其分子机制有待深入研究。未折叠蛋白质反应是低氧适应形成的重要原因。分子伴侣Hsp90 参与底物蛋白折叠与降解的分选，是蛋白质质控中的重要分子，与应激适应关系密切，且已知其多种底物蛋白参与低氧适应调控。本研究在前期已确定Hsp90 与底物蛋白结合、解离受应激强度影响的实验基础上，建立低氧适应细胞模型，应用稳定同位素标记结合固化抗体免疫共沉淀技术，观测Hsp90 与底物蛋白相互作用状态的时序性变化，以生物信息学手段分析Hsp90 对低氧适应信号通路的整合作用。确定了低氧对细胞增殖存在促进作用，并与Hsp90的磷酸化水平升高密切相关；以CRISP-cas9技术建立了Hsp9Oα敲除细胞系；免疫共沉淀分离与Hsp90相互作用的底物蛋白；质谱鉴定候选底物蛋白；生物信息学数据库资源分析可能的交互作用关系；从应激相关Hsp90相互作用底物谱，筛选出与细胞增殖相关的Bclaf1，验证了Hsp90分子伴侣功能对候选底物蛋白Bclaf1稳定性的影响；并进而关注其对低氧相关蛋白HIF1α的转录调控机制。该研究阐明了Hsp90 对低氧预适应信号通路的调控机制，为将与Hsp90 相关的治疗、预防手段应用于低氧相关疾病奠定基础。