Molecular mechanism of the regulation of the mammalian heat shock transcription factor Hsf1

热休克反应是一种普遍的、细胞自主的转录程序。它是由蛋白质稳态失衡引起的，并由热休克转录因子HSF1在所有真核细胞中协调。通过这一功能，HSF1处于许多生理和病理生理过程的中心阶段，如胚胎后发育和衰老、癌症和神经变性。HSF1的活性受内源性应激诱导的构象变化、大量的翻译后修饰以及分子伴侣和其他相互作用的蛋白质控制。HSF1通过释放在热休克基因转录起始点暂停的RNA聚合酶II来发挥其功能。尽管进行了40多年的密集研究，但在分子水平上调节HSF1活性的机制仍然知之甚少。后生动物HSF1由DNA结合区、亮氨酸拉链状七重复区A和B(HR-A/B)、调节区、抑制三聚的第三个七重复区(HR-C)和反式激活区组成。在之前DFG资助的一个项目中，我们证明了HSF1是一个温度传感器，其响应与热应激的严重程度和长度成正比。使用纯化的蛋白质，我们证明了Hsc70和DNAJB1通过HSF1的单体在体外将三聚体HSF1从DNA上解离。Hsc70通过连续的熵拉动循环使HSF1三聚体单体，拉开三链亮氨酸拉链。(1)利用X射线结晶学和低温电子显微镜对HSF1的单体、二聚体和低聚物的结构进行了深入的研究。(2)研制基于生物发光共振能量转移(BRET)的HSF1活性传感器。这种传感器可用于筛选抑制HSF1激活或Hsc70介导的HSF1失活的药物。(3)我们想要分析在三聚化结构域和邻近Hsc70结合位点的翻译后修饰对核心HSF1激活-衰减周期的调制，这是HSF1单体所必需的。(4)通过生化分析和氢交换质谱法研究已报道的HSF1激活剂和抑制剂HSBP1和SSBP1与HSF1的相互作用，以期从分子水平上了解它们的活性。对于AIMS(3)和(4)，我们想要阐明HSF1活性的核心激活-衰减周期如何整合多个输入信号，并根据细胞的需要进行调整。

The heat shock response is a universal, cell-autonomous transcriptional program. It is induced by an imbalance of protein homeostasis and orchestrated in all eukaryotic cells by heat shock transcription factor Hsf1. Through this function Hsf1 is in metazoa at center stage of many physiological and pathophysiological processes like post-embryonic development and aging, cancer and neurodegeneration. The activity of Hsf1 is controlled by intrinsic stress-induced conformational changes, by a large number of post-translational modifications, and by molecular chaperones and other interacting proteins. Hsf1 exerts its function by releasing RNA polymerase II that is paused at the transcription start site in heat shock genes. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation at a molecular level remains poorly understood. Metazoan Hsf1 consists of a DNA binding domain, leucine zipper-like heptad-repeat regions A and B (HR-A/B) functioning as trimerization domain, a regulatory domain, a third heptad repeat region (HR-C), repressing trimerization, and a transactivation domain. In a previously DFG funded project we demonstrated that Hsf1 is a thermosensor, the response of which is proportional to the severity and the length of the heat stress. Using purified proteins, we demonstrated that trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1 by monomerization of Hsf1. Hsc70 monomerizes Hsf1 trimers by successive cycles of entropic pulling, unzipping the three-stranded leucine-zipper.In this project we have four aims. (1) We want to gain insights into the structure of monomeric, dimeric and oligomeric Hsf1 using x-ray crystallography and cryo-electron microscopy. (2) We want to develop Hsf1 activity sensors based on bioluminescence resonance energy transfer (BRET). Such sensors could be used for screening for drugs that inhibit Hsf1 activation or Hsc70-mediated Hsf1 inactivation. (3) We want to analyse the modulation of the core Hsf1 activation-attenuation cycle by posttranslational modifications in the trimerization domain and adjacent to the Hsc70 binding site that is essential for Hsf1 monomerization. (4) We want to study the interaction of the reported Hsf1 activator and inhibitor HSBP1 and SSBP1 with Hsf1 by biochemical assays and hydrogen exchange mass spectrometry to get a molecular understanding of their activity. With aims (3) and (4) we want to elucidate how the core activation-attenuation cycle of Hsf1 activity integrates multiple input signals and is tuned to the needs of the cell.