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: 6639596
• 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/6639596
• 关键词: X ray crystallography; amyotrophic lateral sclerosis; analytical ultracentrifugation; chemical structure; copper; enzyme complex; enzyme structure; gene mutation; 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.

真核铜锌超氧化物歧化酶 (CuZnSOD) 是 32 kDa 同二聚体金属蛋白，可催化超氧自由基转化为分子氧和过氧化氢。这种酶在哺乳动物的脊髓组织和红细胞中特别丰富。 人类 CuZnSOD 中大约 60 种不同的单位点突变分别与遗传性（家族性）肌萎缩侧索硬化症（FALS 或卢伽雷氏病）相关。 该疾病的特征是运动神经元变性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任何一种 FALS CuZnSOD 突变杂合子个体发病的原因。 许多这些人类 FALS 突变体已在实验室中制备，并被发现相对于野生型 CuZnSOD 具有异常的金属结合特性。 FALS 突变蛋白的新特性可能与 FALS 因果关系中毒素功能的获得有关。 最近发现的一类被称为“SOD 铜伴侣”(CCS) 的分子负责从细胞环境中隔离活性铜离子，并将其正确插入新翻译的 SOD 脱辅基蛋白中。 由于 FALS 突变体 SOD 的毒性功能增益似乎是铜介导的，因此寻找铜伴侣的抑制剂可能代表了 FALS 的一种新的治疗途径。 该提案的主要目标是通过完善的 X 射线衍射和分析工具，生成关于人类和酵母 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。超速离心。 具体来说，我们应该：（1）将FALS结构与正常人CuZnSOD和酵母FALS突变体类似物结构进行比较和对比。 (2) 使用来自调谐到铜和锌吸收边缘的同步加速器源的 X 辐射来确定这些离子在重金属化和分离的 FALS CuZnSOD 的金属结合位点中的位置和数量，(3) 阐明了过氧亚硝酸盐与假定的相互作用通过在接触过亚硝酸盐时确定正常和 FALS 突变体 CuZnSOD 蛋白的结构，(4) 确定和分析几种单独的 CCS 分子以及与野生型和 FALS 突变体复合的 CCS 分子的结构CuZnSOD，并使用现代分析超速离心方法阐明了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 分子原因的理解，还将提供有关蛋白质-蛋白质相互作用水平上的铜离子稳态和运输的基本信息。