ZINC DEFICIENT SOD AND NEUROFILAMENT DYSFUNCTION IN ALS

描述：家族性肌萎缩侧索硬化症(FALS)患者 具有35个已知铜锌超氧化物歧化酶(SOD)突变中的任何一个 有85%的几率患上这种致命的神经退行性疾病。 这些超氧化物歧化酶突变体在表达时都会导致运动神经元疾病 转基因小鼠尽管存在正常的补体 野生型超氧化物歧化酶。这些突变体的显性效应表明 超氧化物歧化酶的有害功能而不是简单的丢失 活动。来自该实验室的证据表明，超氧化物歧化酶突变体 锌的亲和力按等级顺序降低，这似乎与 致命性。这些观察结果与以下论证相结合 缺锌超氧化物歧化酶催化过亚硝酸根硝化蛋白质酪氨酸 比含锌的超氧化物歧化酶更有效。此外，神经细丝-L，一个 运动神经元存活的关键蛋白质是超氧化物歧化酶的主要靶点 催化硝化，结合锌，但不结合铜。最后，过氧亚硝酸根 三联体神经丝蛋白的体外治疗组装，推测是通过 在疏水性酪氨酸上引入永久性负电荷。因此，它 假设FALS的选择性运动神经元死亡特征 这是由于SOD突变体异常的金属结合特性造成的 直接或间接导致神经丝的硝化和随后的 抑制功能。提出了以下具体目标 进一步研究超氧化物歧化酶突变体对金属结合的影响 生理相关条件，探索金属可能被 体内缺失突变体，并阐明过氧亚硝酸盐诱导的机制 神经丝功能障碍。具体目标1)评估锌和铜的结合 由SOD突变体、野生型和缺锌形式在 周转和接触一氧化氮和过氧亚硝酸盐。具体目标2) 测量相关蛋白质的锌和铜结合特性，如 神经丝和金属硫蛋白在体外结合金属并测定 这些蛋白质是否可以通过直接改变超氧化物歧化酶的金属含量 通过相互作用或充当金属水槽。具体目标3)确定 单个神经丝蛋白硝化的相对贡献 体外对三联体神经丝组装的抑制作用及其程度 抑制所需的硝化作用。

DESCRIPTION: Familial amyotrophic lateral sclerosis (FALS) patients possessing any one of 35 known mutations to Cu,Zn superoxide dismutase (SOD) stand a >85% chance of developing this fatal neurodegenerative disease. These SOD mutants invariably cause motor neuron disease when expressed in transgenic mice despite the coexistence of the normal complement of wild-type SOD. The dominant effect of these mutants suggests a gained deleterious function rather than simple loss of superoxide dismutase activity. Evidence from this laboratory indicates that SOD mutants have decreased affinity for zinc in a rank order which appears to correlate with virulence. These observations are combined with the demonstration that zinc-deficient SOD catalyzes protein tyrosine nitration by peroxynitrite more efficiently than Zn-containing SOD. Furthermore, neurofilament-L, a protein critical to motor neuron survival, is a major target of SOD catalyzed nitration and binds zinc but not copper. Lastly, peroxynitrite treatment assembly of triplet neurofilament proteins in vitro, presumably by introducing a permanent negative charge on hydrophobic tyrosine. Thus, it is hypothesized that the selective motor neuron death characteristic of FALS results from the aberrant metal binding properties of SOD mutants which directly or indirectly lead to nitration of neurofilaments and subsequent inhibition of function.The following specific aims are proposed to characterize further metal binding-binding by SOD mutants under physiological relevant conditions, explore mechanisms by which metals may be lost from mutants in vivo, and elucidate mechanisms of peroxynitrite-induced neurofilament dysfunction. Specific Aim 1) Assess zinc and copper binding by SOD mutants, wild type, and zinc deficient forms under conditions of turnover and exposure to nitric oxide and peroxynitrite. Specific aim 2) Measure the zinc and copper binding properties of relevant proteins like neurofilaments and metallothioneins which bind metals in vitro and determine whether these proteins can alter the metal content of SODs by direct interaction or by acting as metal sinks. Specific aim 3) Determine the relative contribution of nitration of individual neurofilament proteins to inhibition of triplet neurofilament assembly in vitro and the extent of nitration required for inhibition.