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(SOD1)的突变会导致一种遗传性的致命神经退行性疾病--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，不可溶形式的突变SOD1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，表明SOD1连锁的ALS是一种蛋白质错误折叠和聚集障碍。因此，了解致病突变如何引起易于聚集的SOD1折叠中间体的分子基础是非常有兴趣的。当新生的SOD1多肽被SOD1的铜伴侣蛋白(CCS)翻译后修饰时，折叠途径上的关键一步发生了。CCS是一种辅助蛋白，插入催化铜辅助因子并氧化SOD1亚单位内的二硫键。CCS识别并结合到新生的但不是成熟的SOD1，这表明新翻译的形式存在于不同于成熟形式的构象中。最近的研究表明，从培养细胞和转基因小鼠脊髓中分离出来的致病SOD1蛋白往往是缺乏金属和/或缺乏二硫键的。这些观察结果提示一种假设，即致病突变可能通过阻碍CCS介导的SOD1成熟来提高SOD1折叠中间产物的水平。这个项目中概述的实验旨在探索新生的SOD1的结构和动力学，并检查它与CCS的相互作用。本文所包含的目标的成功完成将解决以下问题：1)什么结构决定因素负责识别新生的SOD1？2)功能性的SOD1/CCS复合体是异二聚体、异四聚体或其他一些更高阶低聚物？3)新生的SOD1/CCS复合体的三维结构细节是什么？4)在没有CCS的情况下，什么因素控制着新生的和成熟的SOD1构象的相互转化？5)ALS突变如何影响新生的和成熟的构象的相互转化？这些问题的答案是设计旨在抑制ALS病理性SOD1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)是一种与衰老相关的进行性神经退行性疾病。在第一次美伊战争中服役的军事人员在回国后患上散发性(非遗传性)肌萎缩侧索硬化症的频率比美国总体人口更高。家族性肌萎缩侧索硬化症(FALS)是一代又一代的遗传疾病，其潜在的遗传缺陷在一些ALS家族被发现编码抗氧化酶铜锌超氧化物歧化酶(SOD1)的基因突变之前是完全未知的。重要的是，散发性和家族性肌萎缩侧索硬化症的临床表现几乎无法区分，这表明这两种疾病的潜在分子病因是相关的。这项建议调查SOD1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以导致适用于散发性疾病的治疗途径，随着疾病的继续老龄化，这两种疾病预计都会对退伍军人群体产生越来越大的影响。

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.