Mechanism of Action of sHSP Chaperones in Protection from Oxidative Stress

描述(申请人提供)：分子伴侣是一种蛋白质，它被细胞压力激活，并帮助防止错误折叠的蛋白质聚集。小分子热休克蛋白伴侣家族(SHSPs)是一类普遍存在的分子伴侣，它们在序列、大小和底物特异性上都是不同的，但具有保守的功能，可以结合部分变性的底物，并以不依赖于ATP的方式阻止它们的不可逆聚集。我这项研究的长期目标是阐明sHSP的伴侣作用机制，并确定sHSP在氧化应激保护中的作用。我将使用蓝藻，聚球藻PCC6803来阐述SHSP在氧化应激过程中作用的基本分子机制。集胞藻只有一个SHSP，Hsp16.6，已被证明是获得耐热性所必需的。重要的是，我最近的数据表明，Hsp16.6也是氧化应激期间生存所必需的。我将使用纯化的重组sHSP进行体外生化研究，以揭示SHSP功能的机制细节，并同时确定在体内氧化应激过程中受到SHSP保护的潜在底物。具体地说，我将1)确定氧化应激是否诱导Hsp16.6发生构象变化，2)确定sHSPs是否能保护酶免受不可逆氧化和失活，3)确定在氧化应激保护过程中与Hsp16.6合作的蛋白质。这些基础研究将更好地了解sHSPs在细胞氧化还原平衡中的作用，以及sHSPs如何影响包括神经退行性疾病、心血管疾病和癌症在内的一系列人类疾病。

DESCRIPTION (provided by applicant): Molecular chaperones are proteins that are activated by cellular stress and assist in preventing the aggregation of misfolded proteins. The small heat shock protein chaperone family (sHSPs) is a ubiquitous class of molecular chaperones that are heterogeneous in sequence, size, and substrate specificity but have a conserved function to bind partially denatured substrates and prevent their irreversible aggregation in an ATP-independent manner. The long term goals of my research are to elucidate the mechanism of sHSP chaperone action and to define the role of sHSPs in protection from oxidative stress. I will address the basic molecular mechanism of sHSP action during oxidative stress using a model organism, the cyanobacterium, Synechocystis PCC 6803. Synechocystis has only one sHSP, Hsp16.6, which has been shown to be required for the acquisition of thermotolerance. Importantly, my recent data demonstrate Hsp16.6 is also necessary for survival during oxidative stress. I will conduct biochemical studies in vitro using purified recombinant sHSPs to uncover mechanistic details of sHSP function and in parallel identify potential substrates protected by an sHSP during oxidative stress in vivo. Specifically I will 1) determine if there are conformational changes in Hsp16.6 induced by oxidative stress, 2) Determine if sHSPs can protect enzymes from irreversible oxidation and inactivation, and 3) Identify proteins that cooperate with Hsp16.6 during protection from oxidative stress. These fundamental studies will provide a better understanding of the role of sHSPs in cellular redox equilibrium and how sHSPs impact a vast spectrum of human diseases including neurodegenerative disorders, cardiovascular disease, and cancer.