X-ray Studies of SOD in Amyotrophic Lateral Sclerosis

描述（由申请方提供）：人铜锌超氧化物歧化酶（SOD 1）是一种32 kDa同型二聚体金属蛋白，可催化超氧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特别丰富。人类SOD 1中大约114个不同的单位点突变与肌萎缩侧索硬化症（ALS，Lou Gehrig病，运动神经元病）的遗传形式有关。该疾病的遗传性（FALS）和散发性（SALS）形式的特征在于由运动神经元变性和死亡引起的进行性瘫痪。现在已经确定，SOD 1-连接的FALS是由细胞毒性性质的获得而不是酶功能的丧失引起的。越来越多的证据表明，毒性来自突变SOD 1蛋白组装成更高级结构（可溶性寡聚体和不溶性聚集体）的能力，这些结构以某种方式干扰神经元细胞机制。使用单晶X射线衍射的成熟工具，我们最近观察到六种不同的金属缺陷型FALS突变体SOD 1蛋白可以形成“淀粉样”纤维，这在某种程度上让人想起在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。致病性SOD 1蛋白能够自我缔合，但正常的未突变的SOD 1蛋白不能。在这个持续的项目中概述的实验旨在探索致病性SOD 1结构，并帮助回答以下问题：1）什么是FALS突变的结构后果，以及这些氨基酸取代如何使分子有毒？2)金属离子的丢失是否在FALSSOD 1致病性中起作用？3)亚基内二硫键的存在与否如何影响致病性SOD 1的结构和生物物理特性？4)我们在X射线研究中观察到的自缔合模式是致病性SOD 1蛋白在活细胞中聚集的基础吗？5)SOD 1蛋白质的哪些结构元件被20 S蛋白酶体识别，从而使它们可以被降解？6)可溶性寡聚体（原纤维）和/或不溶性淀粉样蛋白的致病SOD 1作为蛋白酶体抑制剂？这些问题的答案，如SOD 1连接的FALS的分子理解和治疗药物的设计，旨在抑制聚集过程中所需要的。

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.