Genetic Determinants of Oxygen Toxicity

描述(申请人提供)：含铜和锌的超氧化物歧化酶(SOD1)在抗氧化防御中起着关键作用，但SOD1的突变可通过有毒的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，但一个新出现的主题涉及SOD1错误折叠和形成有毒的SOD1聚集体。这项研究的一个假设是ALS突变体SOD1的命运是由控制多肽翻译后修饰的细胞内因素决定的。我们已经确定了许多这样的因素，包括CCS铜伴侣，一个CCS不依赖于CCS的激活SOD1的途径，以及帮助减少SOD1二硫化物的胞内谷氧还蛋白。这些因素对体内SOD1的确切影响将在当前提案的两个目标中得到解决：AIM1-了解用铜激活人类SOD1的途径：结合遗传学、生物化学和结构分析，将探索人类SOD1如何区分两条获取铜的途径，以及CCS如何通过脯氨酸异构化独特地促进SOD1折叠。目的：了解SOD1相互作用因素对SOD1稳定性和聚集性的影响：酵母和组织培养中的分子遗传学研究将剖析铜激活通路和二硫键还原通路在ALS突变体SOD1折叠和聚集中的相反作用。 在另一项独立但相关的研究中，这项提议还将解决CCS在染色质沉默中的一个前所未有的角色：目的3-了解CCS在SOD1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Sir2的可能机制促进SOD1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SOD1及其金属配位蛋白CCS的生物学提供新的曙光，对疾病具有重要的意义。

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.