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: 6130636
• 负责人: Peter JOHN HART
• 金额: $ 23.39万
• 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/6130636
• 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.

Eucaryotic铜锌超氧化物歧化酶（Cuznsods）是32 kDa同型含量金属蛋白，可促进超氧化物自由基向分子氧和过氧化氢的转化。该酶在脊髓组织和哺乳动物的红细胞中特别丰富。 人类Cuznsod中约有60个不同的单位突变已分别与植物营养性侧面硬化症（Fals或Lou Gehrig病）的遗传（家族性）形式相关。 该疾病的特征是运动神经元变性和死亡引起的进行性瘫痪。 跨牙小鼠，神经元细胞培养和体外研究强烈表明，铜介导的功能毒性增益，而不是超氧化物过度含量的活性丧失是任何一个基因杂合子的疾病发作。 这些人类伪造突变体中的许多是在实验室中制备的，并且发现相对于野生型cuznsod具有异常的金属结合特性。 fals突变蛋白的新特性可能与涉及伪造因果关系的毒素功能的增益有关。 最近发现的一类称为“ SOD铜伴侣”（CCS）的分子类别负责从细胞环境中隔离反应性铜离子，并将其正确插入新翻译的SOD poprototin。 由于伪造突变类草皮功能的毒性增加似乎是铜介导的，因此寻找铜伴侣抑制剂的搜索可能代表了一种新型的伪造治疗途径。 该提案的主要目标是生成有关人和酵母突变体和突变蛋白类似物，它们各自的铜伴侣的三维原子分辨率的结构信息，以及通过X射线衍射和分析的良好工具来生成各自的铜伴侣及其复合物超速离心。 具体而言，我们将：（1）将伪造结构与正常的人类cuznsod和酵母fals fals突变类似物结构进行比较和对比。 （2）使用调谐到铜和锌吸收边缘的同步源的X辐射来确定这些离子在重新测序和脱离的fals的金属结合位点的位置和量正常和虚拟突变体cuznsod蛋白通过暴露于过氧亚硝酸盐时确定其结构，（4）单独确定和分析几种CCS分子的结构，并与野生型和虚拟突变体cuznsods的复合物，并阐明这些分子和这些分子溶液的特性它们在不同条件下使用当代分析超速离心方法的复合物。 这些研究不仅将促进对fal的分子原因的理解，而且还将提供有关铜离子稳态和在蛋白质相互作用水平下运输的基本信息。