Allosteric Mechanism of Hsp70 Molecular Chaperones

描述(申请人提供)：普遍存在的Hsp70分子伴侣家族利用一种看似简单的域间变构机制来完成一系列令人震惊的关键细胞功能。Hsp70促进了新合成蛋白质的折叠，通过挽救错误折叠的蛋白质或将它们引导到降解机制来保护细胞免受压力条件下可能发生的损伤，帮助蛋白质跨膜转移，并促进大分子复合体的组装和拆解。所有这些功能都依赖于Hsp70结合蛋白质底物未折叠区域的能力，以及在变构结合ATP时释放底物的能力。热休克蛋白70因其抗细胞凋亡和致癌活性而成为癌症治疗的潜在靶点。 以及作为与蛋白质错误折叠相关的疾病(囊性纤维化、神经退行性疾病)的靶标，因为它们有能力拯救易于聚集的蛋白质。我们使用大肠杆菌Hsp70，DNAK作为Hsp70变构基本机制的范例，已经显示了变构信号是如何从N-末端结构域的核苷酸结合部位传递到结构域间连接子的，我们有诱人的线索说明这是如何导致C-末端底物结合结构域(SBD)中的主要重组和伴随的底物亲和力降低的。在目标1中，我们将在我们最新进展的基础上，结合新的大蛋白质核磁共振方法和互补的生物物理方法，如脉冲电子自旋共振，将DNAK中的变构信号传递映射到SBD。我们将研究辅助伴侣相互作用如何重塑DNAK变构格局，以及我们新发现的DNAK极端C末端的作用如何影响它与底物的相互作用。在目标2中，通过我们正在进行的DNAK工作收集的方法和知识将应用于四个人类Hsp70，细胞质Hsc70(结构性)和HspA1(诱导)，内质网驻留的Bip和线粒体mtHsp70。这些伴侣机器共享一个共同的机制，但特点是大量的序列多样化，以及随之而来的底物偏好和辅助伴侣的不同。在目标3中，我们将从结构和生物物理上表征Hsp70的小分子调节剂，这些调节剂是由正在探索Hsp70作为药物靶点的合作者提供的。我们将通过核磁共振确定候选小分子与Hsp70的相互作用模式，寻找特定Hsp70选择性的证据以及如何增强选择性，并提供反馈以改善调制器的特性。总体而言，拟议的研究将有助于我们对变构的基本理解，同时为不同Hsp70家族成员的功能和专门化提供见解。这些见解对于最有效地利用Hsp70作为药物靶点至关重要。 与公共健康相关：HSP70分子伴侣在正常生理条件下发挥关键作用，保护细胞免受热休克等应激的影响。Hsp70与几种毁灭性的疾病有关，包括癌症和蛋白质错误折叠疾病，如囊性纤维化、阿尔茨海默氏症和亨廷顿氏症，导致它们成为可能的治疗靶点。这项研究将提供关于Hsp70的基本机制和Hsp70亚家族功能多样性的急需的知识，从而极大地促进其作为药物靶点的使用。

DESCRIPTION (provided by applicant): The ubiquitous Hsp70 family of molecular chaperones utilizes a deceptively simple mechanism of interdomain allostery to accomplish a stunningly broad array of critical cellular functions. Hsp70s facilitate folding of newly synthesizd proteins, protect cells from damage that can occur under stress conditions by either rescuing misfolded proteins or directing them to degradation machinery, assist protein translocation across membranes, and promote assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. Hsp70s have emerged as potential targets for cancer therapeutics because of their anti-apoptotic and tumorigenic activity, and as targets for diseases associated with protein misfolding (cystic fibrosis, neurodegenerative diseases) because of their ability to rescue aggregation-prone proteins. Our work using the E. coli Hsp70, DnaK, as a paradigm for the fundamental mechanism of Hsp70 allostery has shown how allosteric signals are communicated from the nucleotide-binding site of the N-terminal domain to the interdomain linker, and we have tantalizing hints about how this then causes major reorganization and concomitant reduced substrate affinity in the C-terminal substrate-binding domain (SBD). In Aim 1, we will build on our recent progress and map the allosteric signal transmission in DnaK into the SBD, using a combination of novel nuclear magnetic resonance approaches for large proteins, and complementary biophysical methods such as pulsed electron spin resonance. We will study how co-chaperone interactions remodel the DnaK allosteric landscape, and how our newly discovered role for the extreme C-terminus of DnaK may influence its interactions with substrates. In Aim 2, the methods and knowledge gathered through our ongoing work on DnaK will be applied to four human Hsp70s, the cytoplasmic Hsc70 (constitutive) and HspA1 (inducible), the ER-resident BiP, and the mitochondrial mtHsp70. These chaperone machines share a common mechanism but are characterized by substantial sequence diversification and consequent differences in substrate preferences and in co-chaperone partners. In Aim 3, we will structurally and biophysically characterize small molecule modulators of Hsp70s provided to us by collaborators who are exploring Hsp70s as drug targets. We will determine by NMR the mode of interaction of candidate small molecules with Hsp70s, look for evidence of selectivity for particular Hsp70s and how it may be enhanced, and provide feedback to improve characteristics of modulators. Overall, the research proposed will contribute to our fundamental understanding of allostery, while providing insights into function and specialization of different Hsp70 family members. These insights are critical for optimal use of Hsp70s as drug targets. PUBLIC HEALTH RELEVANCE: Hsp70 molecular chaperones play key roles under normal physiological conditions and protect cells against stresses such as heat shock. Hsp70s have been implicated in several devastating diseases including cancers and protein misfolding diseases, such as cystic fibrosis, Alzheimer's, and Huntington's, leading to their emergence as possible therapeutic targets. This research will provide much needed knowledge about basic mechanisms of Hsp70s and functional diversity among Hsp70 subfamilies and thus will greatly facilitate their use as drug targets.