STRUCTURAL CHANGES IN HUMAN SOD1 BY POST-TRANLATIONAL MODIFICATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肌萎缩侧索硬化症是一种以运动神经元死亡为特征的神经退行性疾病。大约20%的遗传性肌萎缩侧索硬化症病例是由编码铜锌超氧化物歧化酶(SOD1)的基因突变引起的。这些突变的一个后果是导致蛋白质的聚集。这些突变发生在这种153个氨基酸的蛋白质的70多个不同的位置，因此很难确定常见的毒性机制。SOD1是一种同源二聚体酶，每个亚基结合两个金属离子，在C57和C146之间存在分子内二硫键。在所有这些翻译后修饰完好无损的情况下，该酶具有高度的热稳定性，熔点超过90℃。最近的生物物理分析表明，酶的早熟形式，既没有金属离子，也没有二硫键，或者两者都缺乏，可能更有可能发生聚集。这种酶的二硫键还原、无金属形式具有边际稳定性，酶的ALS变体甚至可能在生理温度下展开。为了确定SOD1单体形式的结构差异，我们计划用小角X射线散射(SAXS)研究稳定的WT蛋白单体版本和一组选定的ALS变体之间的整体结构差异。这将包括对SOD1的研究，包括多个阶段的翻译后修饰，以及在氧化和还原条件下以及在不同阶段的金属负载下。这一结果有望对我们理解疾病病理的分子机制产生重大影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. Approximately 20% of all inherited cases of ALS are caused by mutations in the gene encoding for Cu, Zn superoxide dismutase (SOD1). One consequence of these mutations is to cause the aggregation of the protein. These mutations occur at over 70 different sites of this 153 amino acid protein, making it difficult to ascertain a common toxic mechanism. SOD1 is a homodimeric enzyme which binds two metal ions per subunit and contains an intramolecular disulfide-bond between C57 and C146. With all of these post-translational modifications intact, the enzyme is highly thermostable, with a melting point over 90 ¿C. Recent biophysical analyses have suggested that premature forms of the enzyme, lacking either the metal ions, the disulfide bond or both, may be more likely candidates for aggregation. The disulfide-reduced, metal-free forms of the enzyme have marginal stability, and ALS-variants of the enzyme may even be unfolded at physiological temperatures. In order to determine structural differences in the monomeric forms of SOD1, we plan to study global structural differences between stable monomeric versions of the WT protein and a selected set of ALS variants with small angle x-ray scattering (SAXS). This will include studies of SOD1, with multiple stages of posttranslational modification and under oxidized and reduced conditions and also under various stages of metal loading. The results are expected to have an significant impact on our understanding of the molecular mechanism of disease pathology