Hsp90α调控Bclaf1蛋白稳定性及其在低氧适应中的作用机制研究

Hypoxia contributes to the extreme environment induced stress response which occupies an important position in special medicine. In order to enhance the survival rate, the organism triggers a variety of physiological responses to promote adaptation to hypoxia. Hsp90 contributes to stress adaptation, and many Hsp90 client proteins are critical for hypoxic adaptation formation. Preliminary experimental results indicated that Hsp90α interacted with Bclaf1 and promoted the stability of Bclaf1. Also, we found that increased expression of Bclaf1 and an enhanced binding of Bclaf1 to Hsp90α occurred in cells under hypoxia. It is known that Bclaf1 plays a vital role in cell survival or proliferation. Whether the Hsp90α-Bclaf1 interaction involves in hypoxia adaptation? In this project, we will establish hypoxic cell line models and hypoxic animal models for parallel comparison, combined stable transfection method and isobaric tags for relative and absolute quantitation (iTRAQ) technique to explore the specific mechanism of Bclaf1. The Hsp90 siRNA and ATPase mutant will be achieved by a plasmid system to analyze how Hsp90 modulates the steady-state of Bclaf1. The immunoprecipitation will be employed to detect the dynamic change of Hsp90α-Bclaf1 interaction upon exposure to hypoxia. We will focus on the role of Hsp90α-Bclaf1 plays in hypoxia adaptation to elucidate the molecular mechanism of hypoxia stress and provide new insight on drug development in hypoxia related diseases caused by special environment.

低氧与极端环境诱发的应激反应密切相关，在特种医学中具有重要地位。机体可通过多种生理反应适应低氧。Hsp90与应激适应相关，其多种底物蛋白参与低氧适应。本课题组前期结果表明，Hsp90α与Bclaf1结合从而促进其蛋白稳定性，低氧刺激Bclaf1上调且Hsp90α-Bclaf1结合增多。已知Bclaf1在细胞增殖、存活中发挥重要作用，Hsp90α-Bclaf1是否参与低氧适应？本研究拟建立低氧细胞和动物模型，采用稳定干扰技术和同位素标记的定量蛋白质组学技术iTRAQ探讨Bclaf1的功能及其机制；以质粒转染技术建立Hsp90α siRNA、ATPase突变体细胞株分析Hsp90α对Bclaf1稳定性的影响；以免疫共沉淀观察低氧应激下Hsp90α-Bclaf1的结合变化，阐明Hsp90α调控Bclaf1稳定性及其在低氧适应中作用和分子机制，为特殊环境所致低氧相关疾病的药物设计和防治提供新思路。

低氧与极端环境诱发的应激反应密切相关，在特种医学中具有重要地位。机体可通过多种生理反应适应低氧。Hsp90与应激适应相关，其多种底物蛋白参与低氧适应。本研究建立低氧细胞模型；以质粒转染技术建立Hsp90α siRNA、ATPase突变体细胞株分析Hsp90α对Bclaf1稳定性的影响；以免疫共沉淀观察低氧应激下Hsp90α-Bclaf1的结合变化，阐明Hsp90α调控Bclaf1稳定性及其在低氧适应中作用和分子机制。本研究通过细胞低氧适应模型下干扰BCLAF1表达和外源性表达BCLAF1结构域缺失体、突变体后，观测HIF-1α的表达量及下游效应分子及细胞低氧相关损伤、适应指标的改变。结果表明，Hsp90α与Bclaf1结合从而促进其蛋白稳定性，低氧刺激Bclaf1上调且Hsp90α-Bclaf1结合增多。已知Bclaf1在细胞增殖、存活中发挥重要作用，Hsp90α-Bclaf1是否参与低氧适应？结果表明，Bcl-2相关转录因子BCLAF1在低氧下高表达，且具有低氧作用时间依赖性，并与HIF1α表达呈正相关。鉴于BCLAF1在细胞内环境稳态及RNA转录和剪切调控发挥关键作用，高度提示其在HIF1α转录及剪切调控中的可能作用。本研究阐明了低氧应激对HSP90-BCLAF1-HIF-1α轴相关的细胞生物学功能的影响，为特殊环境所致低氧相关疾病的药物设计和防治提供新思路。