Nascent SOD1 in Amyotrophic Lateral Sclerosis

描述（由申请人提供）： 人类铜锌超氧化物歧化酶（SOD 1）的突变会导致一种遗传形式的致命性神经退行性疾病肌萎缩侧索硬化症（ALS，Lou Gehrig病，运动神经元病）。随着年龄的增长，不溶性形式的突变体SOD 1逐渐积累在人类ALS患者的神经组织和表达这些多肽的转基因小鼠的脊髓中，这表明SOD 1连接的ALS是一种蛋白质错误折叠和聚集障碍。因此，了解致病突变如何产生易于聚集的SOD 1折叠中间体的分子基础是非常感兴趣的。折叠途径上的关键步骤发生在新生的SOD 1多肽被SOD 1的铜分子伴侣（CCS）后修饰时，CCS是一种插入催化铜辅因子并氧化SOD 1亚基内二硫键的辅助蛋白。CCS识别并结合到新生的，但不是成熟的SOD 1，这表明新翻译的形式存在于不同于成熟形式的构象。最近的研究表明，致病性SOD 1蛋白来自从培养细胞和转基因小鼠脊髓分离的聚集体，往往是金属缺陷和/或缺乏二硫键。这些观察结果表明，致病突变可能通过阻碍CCS介导的SOD 1成熟来提高SOD 1折叠中间体的水平。本项目中概述的实验旨在探索新生SOD 1的结构和动力学，并研究其与CCS的相互作用。成功完成本文所包含的目标将解决以下问题：1）CCS识别新生SOD 1的结构决定因素是什么？2)功能性SOD 1/CCS复合物是异二聚体、异四聚体还是其他更高级的寡聚体？3)新生的SOD 1/CCS复合物在三维空间中的结构细节是什么？4)在没有CCS的情况下，什么因素控制新生和成熟SOD 1构象的相互转化？5)ALS突变如何影响新生和成熟构象的相互转化？对这些问题的回答是设计旨在抑制ALS中致病性SOD 1聚集的治疗剂的先决条件。1 公共卫生相关性： 肌萎缩侧索硬化症（ALS）是一种与衰老过程相关的进行性神经退行性疾病。在第一次美国战争中服役的军人-伊拉克战争，在他们回国后比在整个美国人口中发现的更频繁地发生散发性（非遗传性）ALS。家族性ALS（fALS）在几代人中从父母传给孩子，并且潜在的遗传缺陷是完全未知的，直到发现一些ALS家族在编码抗氧化酶铜锌超氧化物歧化酶（SOD 1）的基因中具有突变。重要的是，散发性和家族性ALS的表现在临床上几乎无法区分，这表明这两种疾病的潜在分子原因是相关的。这项提案调查了与SOD 1相关的ALS，因为了解家族性病例中运动神经元死亡的分子基础可能会导致适用于散发形式的疾病的治疗途径，随着年龄的增长，这两种疾病都将越来越多地影响退伍军人。

DESCRIPTION (provided by applicant): Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease, motor neuron disease). With increasing age, insoluble forms of mutant SOD1 progressively accumulate in neural tissues of human ALS patients and in spinal cords of transgenic mice expressing these polypeptides, suggesting that SOD1- linked ALS is a protein misfolding and aggregation disorder. Understanding the molecular basis for how the pathogenic mutations give rise to SOD1 folding intermediates that are prone to aggregation is therefore of keen interest. A critical step on the folding pathway occurs when the nascent SOD1 polypeptide is posttranslationally modified by the copper chaperone for SOD1 (CCS), a helper protein that inserts the catalytic copper cofactor and oxidizes the SOD1 intrasubunit disulfide bond. CCS recognizes and binds to nascent, but not mature SOD1, suggesting that the newly translated form exists in a conformation distinct from the mature form. Recent studies reveal that pathogenic SOD1 proteins coming from aggregates isolated from cultured cells and from the spinal cords of transgenic mice tend to be metal-deficient and/or lacking the disulfide bond. These observations suggest the hypothesis that the disease-causing mutations may enhance levels of SOD1 folding intermediates by hindering CCS-mediated SOD1 maturation. The experiments outlined in this project are designed to probe the structure and dynamics of nascent SOD1 and to examine its interaction with CCS. Successful completion of the Aims contained herein will address the following questions: 1) What are the structural determinants that are responsible for the recognition of nascent SOD1 by CCS? 2) Is the functional SOD1/CCS complex heterodimeric, heterotetrameric, or some other higher order oligomer? 3) What are the structural details of the nascent SOD1/CCS complex in three dimensions? 4) What factors govern the interconversion of the nascent and mature SOD1 conformations in the absence of CCS? 5) How do the ALS mutations affect the interconversion of the nascent and mature conformations? Answers to these questions are prerequisites for the design of therapeutic agents aimed inhibiting pathogenic SOD1 aggregation in ALS. 1 PUBLIC HEALTH RELEVANCE: Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder linked to the aging process. Military personnel who served in the first U.S.-Iraq war, have developed sporadic (noninherited) ALS upon their return more frequently than found in the United States population as a whole. Familial ALS (fALS) is passed from parent to child over generations, and the underlying genetic defects were entirely unknown until it was discovered that some ALS families possess mutations in the gene encoding the antioxidant enzyme copper- zinc superoxide dismutase (SOD1). Importantly, the manifestations of sporadic and familial ALS are nearly clinically indistinguishable, suggesting that the underlying molecular causes for the two forms of the disease are related. This proposal investigates SOD1-linked ALS because understanding the molecular basis for motor neuron death in familial cases could lead to therapeutic avenues applicable to sporadic forms of the disease, both of which are expected to increasingly affect the veteran population as it continues to age.