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种已知的Cu，Zn超氧化物歧化酶（SOD）突变中的任何一种 有超过85%的机会患上这种致命的神经退行性疾病。 当这些SOD突变体表达在神经细胞中时， 转基因小鼠，尽管存在正常的补体， 野生型SOD。 这些突变体的显性效应表明， 有害的功能，而不是简单的超氧化物歧化酶的损失 活动 来自该实验室的证据表明，SOD突变体 按等级顺序对锌的亲和力降低，这似乎与 毒性。 这些观察结果与证明相结合， 缺锌SOD催化蛋白质酪氨酸被过氧亚硝酸根硝化 比含锌SOD更有效。 此外，神经毒性-L，a 运动神经元存活的关键蛋白，是SOD的主要靶点 催化硝化并结合锌但不结合铜。 最后，过氧亚硝酸盐 在体外处理三联体神经丝蛋白的组装，大概是通过 在疏水酪氨酸上引入永久负电荷。 因此 假设FALS的选择性运动神经元死亡特征 结果从SOD突变体的异常金属结合特性， 直接或间接导致神经丝的硝化， 功能抑制。建议的具体目标如下， 进一步表征SOD突变体在 生理相关条件，探索金属可能被 从突变体在体内丢失，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）评估锌和铜结合 SOD突变体，野生型，和锌缺乏的形式下的条件下， 营业额和暴露于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， 神经丝和金属硫蛋白在体外结合金属并确定 这些蛋白质是否可以通过直接作用改变SOD的金属含量， 相互作用或充当金属汇。 3）确定具体目标 单个神经丝蛋白硝化的相对贡献 体外三重神经丝组装的抑制及其程度 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.