Mechanistic Studies of the Hsp70 Chaperone System

描述(申请人提供)：Hsp70蛋白家族是一类广泛存在的分子伴侣。在所有生物中都有Hsp70家族的多个代表。它们在细胞中的作用包括帮助蛋白质折叠、蛋白质靶向和转位、大分子复合体的组装和拆解、蛋白质到适当的细胞区域的运输，以及将蛋白质输送到蛋白质分解系统以供其处置。Hsp70在阻止蛋白质聚集、变性蛋白质的复性和处理不可逆转的损伤蛋白质中的作用是细胞保护的。在神经系统内，Hsp70有助于防止由淀粉样蛋白聚集体引起的神经退行性变，并在突触小泡循环中发挥基础作用。Hsp70的过表达保护细胞免受各种应激源(如炎症、缺血和自由基氧物种)的破坏。因此，人们对阐明它们的机制很感兴趣。热休克蛋白70循环的基本特征已经知道一段时间了。ATP与Hsp70的ATPase结构域的结合导致蛋白质底物结合域的变构变化，导致底物的释放，从而为新的底物分子的结合奠定了基础。ATP水解和随后的PI释放，导致形成稳定的ADP*Hsp70*底物复合体。ATP水解释放的能量可以被用来驱动蛋白质转运和大分子复合体重构反应。参与HSP70循环的多个步骤受辅伴侣的调节，它可以增加ATP的水解率，并将各种底物输送到HSP70，或者增加ADP/ATP交换的速度。然而，这些过程的确切机制，主要是变构机制，仍然难以捉摸。为了缓解我们理解中的这些不足，我将描述Hsp70在不同变构状态下的结构特征。突变和生化实验将被用来研究观察到的分子间相互作用和从检查这些结构中推断出的机制假说。

DESCRIPTION (provided by applicant): The Hsp70 protein family is a diverse and ubiquitous group of molecular chaperones. There are multiple representatives of the Hsp70 family in all living organisms. Their cellular roles include assistance in protein folding, protein targeting and translocation, the assembly and disassembly of macromolecular complexes, protein transport to appropriate cellular regions, and the delivery of proteins to a proteolytic system for their disposal. The roles of Hsp70s in the prevention of protein aggregation, the refolding of denatured proteins, and the disposal of irreversibly damaged proteins are cytoprotective. Within the nervous system, Hsp70s assist in the prevention of neurodegeneration which results from amyloid aggregates, as well as play a fundamental role in synaptic vesicle recycling. Overexpression of Hsp70s protects cells against the damaging effects of a variety of stressors (i.e. inflammation, ischemia, and free radical oxygen species). Consequently, there is considerable interest in elucidating their mechanism. The basic features of the Hsp70 cycle has been known for some time. ATP binding to the ATPase domain of the Hsp70 induces an allosteric change in the protein substrate binding domain, resulting in the release of substrate, thus setting the stage for the binding of a new substrate molecule. ATP hydrolysis, and the subsequent release of Pi, results in the formation of a stable ADP*Hsp70*substrate complex. The energy released from ATP hydrolysis can be harnessed to drive protein translocation and macromolecular complex remodeling reactions. The multiple steps involved in the Hsp70 cycle are regulated by cochaperones that increase the rate of ATP hydrolysis and deliver various substrates to the Hsp70, or increase the rate ofADP/ATP exchange. However, the precise mechanisms of these processes, chiefly the allosteric mechanism, remain elusive. To alleviate these deficiencies in our understanding, I will characterize the structures of an Hsp70 in distinct, allosteric states. Both mutational and biochemical experiments will be used to investigate the observed intermolecular interactions and mechanistic hypotheses inferred from inspection of these structures.