Structural and Biophysical Properties of Oligermeric SOD1

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis，ALS）是一种致死性、常染色体显性、进行性退行性运动神经系统疾病 神经元[4]。遗传形式，家族性ALS（FALS），占总病例的约5-10%，并且 最好的证明是由于SOD 1的损伤，SOD 1是编码铜锌超氧化物的基因 歧化酶（SOD 1）[5，6]。到目前为止，大约有100种不同的突变，其中大多数导致单个氨基酸 酸取代，已被确定。尽管SOD 1介导的FALS的分子基础仍然存在， 在FALS的脊髓神经元中观察到含有致病性SOD 1蛋白的模糊的聚集体 患者和疾病的转基因小鼠模型[7-9]。这些SOD 1-的形成和/或积累 现在普遍认为，含有聚集体的蛋白质反映了归因于致病性的“毒性功能获得”， SOD 1，尽管它们发挥毒性作用的确切机制仍不清楚。 FALSSOD 1突变在蛋白质的3-D支架上的空间分布是宽的，分为两个 我们称之为“金属结合区”（MBR）和“野生型样”（WTL）突变体[10]。我们以前的 对SOD 1的MBR突变体类的五个成员的晶体学研究表明，它们是金属 不足金属离子的缺乏导致环元件的构象变化，使边缘去保护 蛋白质中的β折叠链。这种保护的丧失反过来又引起了“互动增益”（GOI） 突变的SOD 1二聚体之间的相互作用促进了线状和螺旋状丝状阵列的形成[1]。因此，在本发明中， 金属缺陷酶中的构象重排导致更高级的寡聚体组装 可能代表与FALS相关的SOD 1突变体的共同毒性。 SOD 1的WTL突变体类的一些成员在其脱辅基蛋白形式中是如此不稳定，以至于它们可能 在他们有机会被适当地元化或二聚化之前聚集[11]。但有 其它WTL突变体在其金属化和载脂蛋白形式中与WTSOD 1一样稳定（参见 初步结果，项目1）。对这种明显矛盾的一种可能解释是， WTL突变体SOD 1蛋白由于与蛋白质的异常相互作用而不能在体内适当地金属化。 金属伴侣另一种可能性是这些突变体在体内更容易氧化损伤， 氧化的致病蛋白质在其金属结合能力上受到损害，因此更多地 易于错误折叠和/或聚集。 本研究项目（项目2）的目标是更好地了解 通过使用广泛的互补生物物理方法聚集致病性SOD 1， 将这些信息与我们从合作努力中获得的信息结合起来， 这些蛋白质在体内的聚集。这种对致病性SOD 1聚集的基本理解是一种 这是未来努力获得靶向蛋白质的疾病治疗剂的先决条件 错误折叠和/或蛋白质降解途径。

Amyotrophic lateral sclerosis (ALS) is a fatal, autosomal dominant, progressive degenerative disease of motor neurons [4]. The inherited form, familial ALS (FALS), represents approximately 5-10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper-zinc superoxide dismutase (SOD1) [5, 6]. To date, approximately 100 distinct mutations, most of which result in single amino acid substitutions, have been identified. Although the molecular basis for SOD1-mediated FALS has remained obscure, aggregates containing pathogenic SOD1 proteins are observed in spinal cord neurons of FALS patients and in transgenic mouse models of the disease [7-9]. Formation and/or accumulation of these SOD1 - containing aggregates is now widely believed to reflect the "toxic gain-of-function" ascribed to pathogenic SOD1, although the exact mechanism through which they exert their toxic effects remains unclear. The spatial distribution of FALS SOD1 mutations on the 3-D scaffold of the protein is broad, falling into two categories we term "metal-binding region" (MBR) and "wild type-like" (WTL) mutants [10]. Our previous crystallographic studies on five members of the MBR mutant class of SOD1 reveal that they are metal deficient. The absence of metal ions leads to conformational changes in loop elements that deprotect the edge strands of beta-sheets in the protein. This loss of protection in turn gives rise to a "gain-of-interaction" (GOI) between mutant SOD1 dimers that promotes the formation of linear and helical filamentous arrays [1]. Thus, conformational rearrangement in the metal-deficient enzyme leading to higher order oligomeric assemblies could represent the toxic property common to mutants of SOD1 linked to FALS. Some members of the WTL mutant class of SOD1 are so destabilized in their apoprotein forms that they may aggregate before they ever have a chance to be metaHated or dimerize properly [11]. However, there are other WTL mutants that are just as stable as WT SOD1 in both their metallated and apo- forms (see Preliminary Results, Project 1). One possible explanation for this apparent contradiction is that the latter WTL mutant SOD1 proteins are not properly metallated in vivo due to abnormal interactions with metallochaperones. Another possibility is that these mutants are more easily oxidatively damaged in vivo and that the oxidized pathogenic protein suffers an impairment in its metal binding ability and thus is more susceptible to misfolding and/or aggregation. The goals of this research project (Project 2) are to derive a better understanding of the mechanisms of aggregation of pathogenic SOD1 through the use of a wide range of complementary biophysical methods and to couple this information with that gained from our collaborative efforts to probe the determinants of aggregation of these proteins in vivo. This fundamental understanding of pathogenic SOD1 aggregation is a prerequisite for future efforts aimed at obtaining therapeutic agents for the disease that target protein misfolding and/or protein degradation pathways.