X-RAY STUDIES OF SOD IN AMYOTROPHIC LATERAL SCLEROSIS

SOD 在肌萎缩侧索硬化症中的 X 射线研究

• 批准号: 6394222
• 负责人: Peter JOHN HART
• 金额: $ 21.68万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-05 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6394222
• 关键词: X ray crystallography; amyotrophic lateral sclerosis; analytical ultracentrifugation; chemical structure; copper; enzyme complex; enzyme structure; molecular chaperones; molecular pathology; peroxynitrites; structural biology; superoxide dismutase; zinc

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.

真核生物铜锌超氧化物歧化酶（cuznsod）是一种32 kDa的二聚体金属蛋白，可催化超氧化物自由基转化为分子氧和过氧化氢。这种酶在哺乳动物的脊髓组织和红细胞中含量尤其丰富。人类CuZnSOD中大约有60种不同的单位点突变分别与遗传性（家族性）肌萎缩性侧索硬化症（FALS或Lou Gehrig's病）有关。该病的特点是运动神经元退化和死亡导致进行性瘫痪。转基因小鼠、神经元细胞培养和体外研究强烈表明，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FALS CuZnSOD突变杂合个体发病的原因。许多这些人类FALS突变体已经在实验室中制备，并且已经发现与野生型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病因中涉及的毒素功能的获得有关。最近发现的一类分子被称为“SOD铜伴侣”（CCS），它们负责将活性铜离子从细胞环境中隔离出来，并将其正确插入新翻译的SOD载脂蛋白中。由于FALS突变sod的功能毒性增加似乎是铜介导的，因此寻找铜伴侣抑制剂可能代表了FALS的新治疗途径。本研究的主要目的是通过成熟的x射线衍射和分析超离心工具，生成人类和酵母FALS突变体和突变蛋白类似物、它们各自的铜伴侣及其复合物的三维原子分辨率结构信息。具体而言，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缘的同步子源的X射线来确定这些离子在再金属化和分离的FALS CuZnSOD的金属结合位点的位置和数量；(3)通过测定暴露于过氧亚硝酸盐时正常和FALS突变CuZnSOD蛋白的结构，阐明了假设的过氧亚硝酸盐与正常和突变CuZnSOD蛋白的相互作用；(4)测定和分析几种CCS分子单独和与野生型和FALS突变型cuznsod复合物的结构，并利用现代分析超离心方法阐明这些分子及其复合物在不同条件下的溶液性质。这些研究不仅将促进对FALS的分子原因的理解，而且将在蛋白质-蛋白质相互作用水平上提供铜离子稳态和运输的基础信息。