X-RAY STUDIES OF SOD IN AMYOTROPHIC LATERAL SCLEROSIS

真核铜锌超氧化物歧化酶(CuZnSOD)是一种32 kDa的同源二聚体金属蛋白，能催化超氧阴离子自由基转化为分子氧和过氧化氢。这种酶在哺乳动物的脊椎组织和红细胞中尤其丰富。大约60个不同的人类CuZnSOD单点突变分别与遗传性(家族性)肌萎缩侧索硬化症(FALS或Lou Gehrig病)有关。这种疾病的特征是运动神经元退化和死亡导致的进行性瘫痪。转基因小鼠、神经细胞培养和体外研究有力地表明，铜介导的毒性功能获得而不是超氧化物歧化活性的丧失是导致任何一种FAL CuZnSOD突变杂合子个体发病的原因。许多这些人类fals突变体已经在实验室中制备，并被发现与野生型CuZnSOD相比具有异常的金属结合特性。FALS突变蛋白的新特性可能与FALS致病相关毒素功能的获得有关。最近发现的一类分子被称为超氧化物歧化酶的铜伴侣(CCS)，负责将活性铜离子从细胞环境中隔离出来，并将其正确地插入到新翻译的超氧化物歧化酶脱辅蛋白中。由于FALS突变超氧化物歧化酶的毒性功能获得似乎是铜介导的，寻找铜伴侣蛋白的抑制剂可能是FALS的一种新的治疗途径。这项建议的主要目标是通过成熟的X射线衍射和分析超速离心工具，生成关于人和酵母FALS突变体和突变蛋白类似物、它们各自的铜伴侣及其络合物的三维、原子分辨率结构信息。具体地说，我们将：(1)将FALS结构与正常人CuZnSOD和酵母FALS突变类似结构进行比较和对比。(2)利用调谐到铜和锌吸收边的同步子源的X射线辐射来确定这些离子在重金属和As隔离的FeS CuZnSODS金属结合部位的位置和数量；(3)通过确定过氧亚硝酸根暴露于正常和fals突变CuZnSOD时的结构来阐明过氧亚硝酸根与正常和fals突变CuZnSOD蛋白的假设相互作用；(4)测定和分析几种CCS分子单独以及与野生型和fals突变CuZnSODS形成的络合物的结构，并用当代分析超速离心法阐明这些分子及其络合物在不同条件下的溶液性质。这些研究不仅将促进对FALS分子原因的理解，而且将在蛋白质-蛋白质相互作用的水平上为铜离子的动态平衡和转运提供基础信息。

Eucaryotic copper-zinc superoxide dismutases (CuZnSODs) are 32 kDa homodimeric metalloproteins that catalyze the conversion of superoxide radical to molecular oxygen and hydrogen peroxide. The enzyme is particularly abundant in spinal tissue and red blood cells in mammals. Approximately 60 different single site mutations in human CuZnSOD have individually been linked to an inherited (familial) form of amyotrophic lateral sclerosis (FALS or Lou Gehrig's disease). The disease is characterized by progressive paralysis resulting from motor neuron degeneration and death. Trangenic mouse, neuronal cell culture, and in-vitro studies suggest strongly that a copper-mediated toxic gain of function instead of a loss of superoxide disproportionation activity is responsible for disease onset in individuals heterozygous for any one of the FALS CuZnSOD mutations. Many of these human FALS mutants have been prepared in the laboratory and have been found to have aberrant metal binding properties relative to wild type CuZnSOD. The new properties of the FALS mutant proteins will likely be linked to the gain of the toxin function involved in FALS causation. A recently discovered class of molecules termed "copper chaperones for SOD" (CCS) are responsible for sequestering reactive copper ion from the cellular environment and inserting it correctly into newly translated SOD apoprotein. Because the toxic gain of function of FALS mutant SODs appears to be copper-mediated, a search for inhibitors of the copper chaperone may represent a novel therapeutic avenue for FALS. The primary objectives of this proposal are to generate three-dimensional, atomic resolution structural information on the human and yeast FALS mutants and mutant protein analogs, their respective copper chaperones, and their complexes via the well-established tools of X-ray diffraction and analytical ultracentrifugation. Specifically, we shall: (1) compare and contrast the FALS structures with normal human CuZnSOD and yeast FALS mutant analog structures. (2) use X- radiation from synchroton sources tuned to copper and zinc absorption edges to determine the location and amount of these ions in the metal binding sites of remetallated and as-isolated FALS CuZnSODs, (3) illuminate the postulated interaction of peroxynitrite with normal and FALS mutant CuZnSOD proteins by determining their structures when exposed to peroxynitrite, (4) determine and analyze the structures of several species of CCS molecules alone and in complex with wild type and FALS mutant CuZnSODs, and elucidate the solution properties of these molecules and their complexes under different conditions using contemporary analytical ultracentrifugation methods. These studies will not only promote understanding of the molecular causes of FALS, but also will provide fundamental information on copper ion homeostasis and trafficking at the level of protein- protein interactions.