X-RAY STUDIES OF SOD IN AMYOTROPHIC LATERAL SCLEROSIS

真核生物铜锌超氧化物歧化酶（CuZnSODs）是一种分子量为32 kDa的同源二聚体金属蛋白，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中约有60种不同的单位点突变分别与肌萎缩性侧索硬化症（ALS或Lou Gehrig病）的遗传（家族）形式有关。 该疾病的特征是由运动神经元变性和死亡引起的进行性瘫痪。 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丧失是导致任何一种FALSCuZnSOD突变杂合子个体发病的原因。 许多这些人的FALS突变体已在实验室中制备，并已被发现具有异常的金属结合性能相对于野生型CuZnSOD。 新的属性的FALS突变体蛋白质将可能与获得的毒素功能参与的FALS的因果关系。 最近发现的一类称为“SOD铜分子伴侣”（CCS）的分子负责从细胞环境中螯合活性铜离子，并将其正确插入新翻译的SOD脱辅基蛋白。 由于功能的毒性增益的FALS突变SOD似乎是铜介导的，寻找铜伴侣的抑制剂可能代表一种新的治疗途径的FALS。 该提案的主要目标是通过X射线衍射和分析超离心的成熟工具，生成关于人和酵母的FALS突变体和突变蛋白类似物、其各自的铜分子伴侣及其复合物的三维、原子分辨率的结构信息。 具体而言，我们将：（1）比较和对比的FALS结构与正常人CuZnSOD和酵母的FALS突变体类似物结构。(2)使用来自调谐到铜和锌吸收边缘的同步辐射源的X射线来确定这些离子在再金属化和分离的FALSCuZnSOD的金属结合位点中的位置和量，（3）通过确定当暴露于过氧亚硝酸盐时它们的结构来阐明过氧亚硝酸盐与正常和FALSCuZnSOD蛋白的假定相互作用，（4）利用现代分析超离心方法，对几种CCS分子及其与野生型和FALS突变型CuZnSODs复合物的结构进行了测定和分析，并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，而且将在蛋白质-蛋白质相互作用水平上提供关于铜离子稳态和运输的基础信息。

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.