人热休克因子hHSF1与hHSF2的三维结构及其功能关系研究

The heat shock response (HSR) is an ordered genetic response to diverse environmental and physiological stressors that results in the immediate induction of genes encoding molecular chaperones, proteases, and other proteins essential for protection and recovery from cellular damage associated with the increase of misfolded, unfolded and aggregated proteins. The list of "stressors" that activate transcription of these heat shock genes is large and includes various acute and chronic conditions such as elevated temperatures, heavy metals, small molecule chemical toxicants, infection, and oxidative stress. Mutations and environmental influences including inflammation, ischemia, tissue wounding and repair, cancer, and neurodegenerative diseases are also associated with the aberrant expression of heat shock proteins. In eukaryotes, HSR is mediated by a positive control upon the hsp genes from specific activator proteins, called heat shock transcription factors, HSFs. Mammals express four members of the HSF familiy, HSF 1-4, which share well-conserved DNA-binding and trimerization domains. Our aim in this project is to explore the molecular basis of mechanisms that control HSF activity and protein homeostasis networks in cells from the perspective of structural biology. Our proposal encompasses the determination of the three-dimensional structures of human HSF1 and HSF2 with the techniques of X-ray crystallography; the identification of structural motifs that play crucial roles in determining or affecting the binding preferences on DNA sequences, homo- or hetero-trimerization, post-translational modifications and transactivation competence of HSFs. Our long-term goal is also performing and in silico screening or ab initio design of small molecule ligands which may serve as potential activators or inhibitors of HSF activity on the basis of structure-analytic results. The research proposed in this application will help us to establish the molecular nature and biological significance of the interaction between HSF1 and HSF2. The obtained results will have important ramifications in the design of pharmacological approaches to either stimulate or inhibit HSF-mediated transcription, depending on the medical problem, i.e. enhanced HSF activity in cancer versus impaired HSF activity in neurodegenerative disorders. The ultimate goal of the project, in short, is to obtain a comprehensive picture at a molecular level of how HSF1 and HSF2 work cooperatively as the master regulators for HSR, and implications on pre-clinical evaluations targeting on these transcription factors.

热休克转录因子（HSF）是调控细胞应激反应、发育与分化、细胞代谢与衰老等重要生理活动的核心因子，同时又与多种癌症及早老型痴呆症、帕金森综合症等神经退休性疾病的发生密切相关。真核细胞HSF的激活是一个多层次调控的复杂过程，其中许多细节与分子机制目前仍不清楚。当前的研究热点包括：1，HSF的上游调控因子与作用机理；2，HSF被激活形成三体的结构基础与相关分子机制；3，决定HSF下游调控对象特异性的结构元件。这些问题的阐明都依赖于HSF分子三维结构的解析。本课题的目标是通过解析此家族两个重要成员HSF1与HSF2的全长晶体结构，以及DNA结合结构域与各自底物DNA的复合物结构，在结构分析的基础上，结合其他实验技术，深入探讨结构与功能关系，揭示HSF活化过程中若干重要分子事件发生的结构基础，阐释可能分子机制。研究结果不仅将为HSF的基础研究领域带来突破性进展，还可为多种疾病相关医学研究提供新思路。

热休克转录因子（HSF）是调控细胞应激反应、发育与分化、细胞代谢与衰老等重要生理活动的核心因子，同时又与多种癌症及早老型痴呆症、帕金森综合症等神经退休性疾病的发生密切相关。人源热休克因子（HSF）家族共包括4个成员，HSF1-4。其中HSF1被认为是参与热休克反应（HSR）调控的主要转录因子，而HSF2则主要在发育及细胞分化中发挥调控作用。二者序列同源，结构相似，在结合DNA时都识别hsp基因启动子区的热休克元件（HSE）。但由于缺乏三维结构信息，HSF1／HSF2如何专一识别并结合HSE的分子机制并不清楚。此外，这两个转录因子在细胞内经历了不同的翻译后修饰，也由此产生不同的生物学效应，但其背后的结构基础也尚未明确。在本项目中，我们通过解析HSF1与HSF2蛋白N-端DNA结合结构域（DBD）的晶体结构，以及它们与各自所偏爱结合的双链DNA的复合物晶体结构，从结构生物学的角度解答以上科学问题。并在高分辨率晶体结构的基础上，通过分子对接与分子动力学模拟揭示了两个转录因子结合DNA底物时的分子构象变化，由此所得结论可以很好地解释在多个分子与细胞生物学实验中所观察到的现象。这对于了解HSF1/HSF2在HSR调控中扮演的不同角色将具有重要提示意义。