Genetic Determinants of Oxygen Toxicity

描述（由申请人提供）：含铜和锌的超氧化物歧化酶（SOD 1）在抗氧化防御中起关键作用，但SOD 1突变可通过毒性功能获得导致肌萎缩侧索硬化症（ALS）或Lou Gehrigs病。潜在的机制尚不清楚，但一个新的主题涉及SOD 1错误折叠和有毒SOD 1聚集体的形成。本研究的一个假设是，ALS突变体SOD 1的命运是由细胞内因子控制的多肽的翻译后修饰。我们已经确定了一些这样的因素，包括CCS铜分子伴侣，一个CCS独立的途径激活SOD 1，和细胞溶质谷氧还蛋白，帮助减少SOD 1二硫化物。这些因素在体内如何影响SOD 1将在当前提案的两个目标中得到解决：目标1-了解铜激活人类SOD 1的途径：遗传学，生物化学和结构分析的组合将探索人类SOD 1如何区分两种获得铜的途径以及CCS如何独特地促进SOD 1通过脯氨酸异构化折叠。目的2-了解SOD 1相互作用因子对SOD 1稳定性和聚集的影响：酵母和组织培养中的分子遗传学研究将剖析铜激活途径与ALS突变体SOD 1折叠和聚集中二硫键还原途径的相反作用。 在一个单独但相关的研究中，该提案还将解决CCS在染色质沉默中前所未有的作用：目的3-了解CCS在SOD 1激活和染色质沉默中的双重作用。酵母遗传学和生物化学将解决CCS如何通过涉及抗衰老因子SIR 2的可能机制促进SOD 1激活和染色质沉默。 总之，这些在酵母和哺乳动物细胞中的研究有望为SOD 1及其金属伴侣CCS的生物学提供新的线索，对疾病具有重要意义。

DESCRIPTION (provided by applicant): Copper and zinc containing superoxide dismutase (SOD1) plays a critical role in anti-oxidant defense, yet mutations in SOD1 can lead to amyotrophic lateral sclerosis (ALS), or Lou Gehrigs disease, through a toxic gain of function. The underlying mechanism remains unclear, but an emerging theme involves SOD1 misfolding and formation of toxic SOD1 aggregates. A hypothesis of this research is that the fate of ALS mutant SOD1 is governed by intracellular factors that control post-translational modification of the polypeptide. We have identified a number of such factors, including the CCS copper chaperone, a CCS-independent pathway for activating SOD1, and cytosolic glutaredoxins that help reduce the SOD1 disulfide. Precisely how these factors impact on SOD1 in vivo will be addressed in two Aims of the current proposal: Aim1-To understand the pathways for activating human SOD1 with copper: A combination of genetics, biochemistry and structural analyses will explore how human SOD1 discerns between two pathways for acquiring copper and how CCS may uniquely promote SOD1 folding through proline isomerization. Aim2-To understand the impact of SOD1 interacting factors on the stability and aggregation of SOD1: Molecular genetic studies in yeast and tissue culture will dissect the opposing roles of the copper activation pathways versus the disulfide reduction pathways in the folding and aggregation of ALS mutant SOD1. In a separate but related line of research, this proposal will also address an unprecedented role for CCS in chromatin silencing: Aim 3-To understand the dual roles of CCS in SOD1 activation and chromatin silencing. Yeast genetics and biochemistry will address how CCS can promote both SOD1 activation and chromatin silencing through a possible mechanism involving the anti-aging factor SIR2. Together, these studies in both yeast and mammalian cells promise to shed new light into the biology of SOD1 and its metallochaperone CCS with important implications to disease.