X-ray Studies of SOD in Amyotrophic Lateral Sclerosis

描述（由申请人提供）：人铜锌超氧化物歧化酶（SOD1）是一种32 kDa的同型二聚体金属蛋白，可催化超氧化物自由基转化为分子氧和过氧化氢。这种酶在红细胞和脊髓组织中尤其丰富。人类SOD1中大约114种不同的单位点突变与遗传性肌萎缩性侧索硬化症（ALS, Lou Gehrig's病，运动神经元病）有关。遗传性（FALS）和散发性（SALS）形式的疾病都以运动神经元退化和死亡导致的进行性瘫痪为特征。现在已经确定，sod1相关的FALS是由于细胞毒性的增加而不是酶功能的丧失。越来越多的证据表明，这种毒性来自于突变SOD1蛋白组装成高阶结构（可溶低聚物和不可溶聚集体）的能力，这种能力以某种方式干扰了神经元细胞机制。使用成熟的单晶x射线衍射工具，我们最近观察到六种不同的缺金属FALS突变SOD1蛋白可以形成“淀粉样”纤维，这有点让人想起其他神经退行性疾病（如阿尔茨海默病和帕金森病）中看到的纤维类型。致病的SOD1蛋白能够自我结合，而正常的、未突变的SOD1蛋白则不能。这个正在进行的项目中概述的实验旨在探测致病SOD1结构，并帮助回答以下问题：1)FALS突变的结构后果是什么？这些氨基酸取代如何使分子毒性？2)金属离子的丢失是否在FALS SOD1致病性中起作用？3)亚基内二硫键的存在或缺失如何影响致病性SOD1的结构和生物物理性质？4)我们在x射线研究中观察到的自关联模式是致病SOD1蛋白在活细胞中聚集的基础吗？5) SOD1蛋白被20s蛋白酶体识别并被降解的结构元件是什么？6)致病性SOD1的可溶性低聚物（原纤维）和/或不溶性淀粉样蛋白是否可以作为蛋白酶体抑制剂？这些问题的答案对于SOD1相关FALS的分子理解和旨在抑制聚集过程的治疗剂的设计是必需的。

DESCRIPTION (provided by applicant): Human copper-zinc superoxide dismutase (SOD1) is a 32 kDa homodimeric metalloprotein that catalyzes the conversion of superoxide radical into molecular oxygen and hydrogen peroxide. The enzyme is particularly abundant in red blood cells and spinal tissue. Approximately 114 different single site mutations in human SOD1 have been linked to an inherited form of amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease, motor neuron disease). Both the inherited (FALS) and sporadic (SALS) forms of the disease are characterized by progressive paralysis resulting from motor neuron degeneration and death. It is now established that SOD1-linked FALS results from the gain of a cytotoxic property and not a loss of enzymatic function. Evidence is accumulating that the toxic property comes from the ability of the mutant SOD1 proteins to assemble into higher order structures (soluble oligomers and insoluble aggregates) that somehow interfere with the neuronal cellular machinery. Using the well established tools of single crystal X-ray diffraction, we recently observed that six different metal-deficient FALS mutant SOD1 proteins can form "amyloid-like" fibers that are somewhat reminiscent of the types of fibers seen in other neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The pathogenic SOD1 proteins are able to self- associate but the normal, unmutated SOD1 proteins cannot. The experiments outlined in this continuing project are designed to probe pathogenic SOD1 structure and to help answer the following questions: 1) What are the structural consequences of the FALS mutations and how do these amino acid substitutions render the molecule toxic? 2) Does the loss of metal ions play a role in FALS SOD1 pathogenicity? 3) How does the presence or absence of the intrasubunit disulfide bond influence the structural and biophysical properties of pathogenic SOD1? 4) Is the mode of self-association we observe in X-ray studies the basis for how the pathogenic SOD1 proteins aggregate in living cells? 5) What are the structural elements of SOD1 proteins that are recognized by the 20 S proteasome so that they may be degraded? 6) Could soluble oligomers (protofibrils) and/or insoluble amyloids of pathogenic SOD1 act as proteasomal inhibitors? Answers to questions such as these are required for a molecular understanding of SOD1 linked FALS and for the design of therapeutic agents aimed inhibiting the aggregation process.