Mechanistic Studies of the Hsp70 Chaperone System

描述（由申请人提供）：Hsp 70蛋白家族是一组多样且普遍存在的分子伴侣。Hsp 70家族在所有生物体中都有多种代表。它们的细胞作用包括协助蛋白质折叠、蛋白质靶向和易位、大分子复合物的组装和分解、蛋白质转运至适当的细胞区域以及将蛋白质递送至蛋白水解系统以进行处置。热休克蛋白70在防止蛋白质聚集、变性蛋白质重折叠和处理不可逆损伤蛋白质中的作用是细胞保护性的。在神经系统内，Hsp 70有助于预防由淀粉样蛋白聚集体引起的神经变性，以及在突触囊泡再循环中发挥重要作用。Hsp 70 s的过表达保护细胞免受各种应激源（即炎症、缺血和自由基氧物种）的破坏作用。因此，有相当大的兴趣在阐明其机制。热休克蛋白70循环的基本特征已经知道了一段时间。ATP与Hsp 70的ATP酶结构域的结合诱导蛋白质底物结合结构域的变构变化，导致底物的释放，从而为新底物分子的结合奠定基础。ATP水解和随后的Pi释放导致形成稳定的ADP* Hsp 70 * 底物复合物。ATP水解释放的能量可用于驱动蛋白质移位和大分子复合物重塑反应。参与Hsp 70循环的多个步骤由辅伴侣调节，辅伴侣增加ATP水解速率并将各种底物递送到Hsp 70，或增加ADP/ATP交换速率。然而，这些过程的确切机制，主要是变构机制，仍然难以捉摸。为了减轻这些缺陷，在我们的理解，我将表征热休克蛋白70在不同的，变构状态的结构。突变和生物化学实验将被用来研究观察到的分子间相互作用和从这些结构的检查推断的机制假说。

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.