MUTANT SUPEROXIDE DISMUTASES--PROPERTIES AND FUNCTION

突变的超氧化物歧化酶——特性和功能

• 批准号: 2416242
• 负责人: LESLIE A. SHINOBU
• 金额: $ 8.97万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2416242
• 关键词: SDS polyacrylamide gel electrophoresis; acid base balance; active sites; chemical association; chemical binding; copper; disease /disorder model; enzyme activity; enzyme mechanism; gene mutation; genetically modified animals; high performance liquid chromatography; histidine; hydrogen peroxide; immunocytochemistry; laboratory mouse; light microscopy; mutant; nitrites; oxidative stress; protein purification; proteolysis; superoxide dismutase; thermostability; tissue /cell culture

The presence of pathogenic mutations in the gene coding for the copper- zinc-containing superoxide dismutase (CuZnSOD) on chromosome 21 in a subset of patients with amyotrophic lateral sclerosis (ALS) has now been firmly established. Over the last year, it has become increasingly clear that the role of mutant CuZnSOD in this disease is not directly related to its function as a superoxide dismutator. Data garnered from the expression of the cDNA for mutant SODs in various cell lines and transgenic mice support the theory that a gain of an as yet unidentified adverse function triggers motor neuron cell death. Although very recent work suggests that alterations in the tertiary structure of the enzyme play a critical role in targeted cell death the salient biochemical properties of the mutant SODs have not yet been well characterized. In this research proposal 7 mutant and the wild-type human CuZnSOD cDNA clones are overexpressed in a baculovirus/Sf21 insect cell system. In Part I, various aspects of their thermodynamic stability and chemical reactivity are defined, i.e.tendency to a) denature in the face of changing pH, temperature guanidine hydrochloride or SDS. b) lose active site copper and c) dismutate superoxide. In Part II, mechanisms for the relative instability of these enzymes are probed. Rates and extent of auto-oxidation of histidine. and 3-nitrotyrosine and dityrosine formation on exposure to hydrogen peroxide or peroxynitrite are determined. This information is compared to the relative susceptibility of these proteins to proteolytic digestion before and after oxidative modification, anti to their half-lives. In Part III patterns of oxidative damage are discerned in vitro in target molecules (DNA albumin neurofilaments) and in vivo in transgenic mice overexpressing mutant CuZnSO. This is accomplished by quantitating well accepted markers of oxidative damage (3-nitrotyrosine dityrosine, protein carbonyls. and 2-oxo-histidine). These studies will be among the first to link known mutations in CuZnSOD with defined changes in the physical and chemical properties, combined with detailed analysis of associated patterns of oxidative stress in the brain and spinal cord, and clinical information such as age of onset severity of disease, time to death.

编码铜的基因中存在致病突变 含锌的超氧化物歧化酶（cuznsod）在21号染色体上 肌萎缩性外侧硬化症患者（ALS）的子集现已 牢固确立。 在过去的一年中，越来越清楚 突变库兹诺德在这种疾病中的作用与 它作为超氧化物拆卸器的功能。 从中获得的数据 在各种细胞系中突变草皮的cDNA表达， 转基因小鼠支持以下理论： 不良功能会触发运动神经元细胞死亡。虽然很新 工作表明酶的三级结构的改变 在靶向细胞死亡中发挥关键作用 突变草皮的性质尚未得到很好的特征。 在这项研究建议中7突变体和野生型人类c.nsod cDNA 克隆在杆状病毒/SF21昆虫细胞系统中过表达。在 第一部分，其热力学稳定性和化学的各个方面 反应性是定义的，即a）面对 更改pH，温度鸟嘌呤盐酸或SDS。 b）失去活跃 现场铜和c）拆卸超氧化物。在第二部分中， 探测这些酶的相对不稳定。速率和程度 组氨酸的自身氧化。和3-硝基酪氨酸和丁乙氨酸的形成 暴露于过氧化氢或过氧亚硝酸盐时。这 将信息与这些蛋白质的相对敏感性进行比较 在氧化修饰之前和之后进行蛋白水解消化，抗 他们的半衰期。在第三部分中，氧化损伤的模式被辨别 靶分子（DNA白蛋白神经丝）和体内体外体外体外体外 过表达突变体的转基因小鼠。这是由 定量氧化损伤的良好接受标记（3-硝基酪氨酸 DITYROSINE，蛋白羰基。和2-氧基 - 抗二碱）。这些研究将是 在第一个将Cuznsod中已知突变与定义的变化联系起来的 物理和化学特性，结合了对 相关的大脑和脊髓中氧化应激的模式，以及 临床信息，例如疾病的发病严重程度，时间 死亡。