Structural and Biophysical Properties of Oligermeric SOD1

寡聚 SOD1 的结构和生物物理特性

基本信息

批准号：
7800916
负责人：
P J HART
金额：
$ 34.59万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7800916
关键词：
3-Dimensional Amino Acid Substitution Amyotrophic Lateral Sclerosis Animals Apoproteins Binding Binding Sites Biological Assay C-terminal Categories Cell Aggregation Cell Culture Techniques Cell model Chemistry Congo Red Copper DNA Sequence Rearrangement Degenerative Disorder Degradation Pathway Disease Dissociation Electrostatics Elements Embryo Engineering Enzymes Familial Amyotrophic Lateral Sclerosis Filament Future Goals Human Hydrogen Peroxide Impairment In Vitro Indium Inherited Ions Laboratories Lead Lesion Link Location Maps Mass Spectrum Analysis Mediating Metal Binding Site Metals Methods Modeling Modification Molecular Molecular Probes Monitor Motor Neurons Mus Mutation Neurons Patients Peroxidases Phosphines Process Production Property Protein Engineering Proteins Reaction Research Project Grants Roentgen Rays Role SOD1 gene Scaffolding Protein Series Site Solubility Solutions Spatial Distribution Spinal Cord Stress Structure Study Section Superoxides Surface Plasmon Resonance Testing Therapeutic Agents Toxic effect Transgenic Animals Transgenic Mice Variant Work X ray diffraction analysis X-Ray Diffraction analytical ultracentrifugation base beta pleated sheet copper zinc superoxide dismutase crosslink deletion analysis design dimer disulfide bond falls gain of function guanidinium in vitro testing in vivo interest kidney cell light scattering member monomer mouse model mutant neurotoxicity novel oxidation oxidative damage prevent programs protein aggregation protein degradation protein misfolding protein oligomer reaction rate research study three dimensional structure tissue culture tool

项目摘要

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.

肌萎缩侧索硬化症 (ALS) 是一种致命的、常染色体显性遗传、进行性运动神经退行性疾病神经元[4]。遗传性家族性 ALS (FALS) 约占总病例的 5-10%，并且其中最有据可查的是 SOD1 的损伤，SOD1 是编码铜锌超氧化物的基因歧化酶 (SOD1) [5, 6]。迄今为止，大约有 100 个不同的突变，其中大多数导致单氨基酸酸取代，已被鉴定。尽管 SOD1 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 SOD1 蛋白的模糊聚集体患者和该疾病的转基因小鼠模型中[7-9]。这些 SOD1 的形成和/或积累 - 现在人们普遍认为含有聚集体反映了致病性的“毒性功能获得” SOD1，尽管它们发挥毒性作用的确切机制仍不清楚。 FALS SOD1 突变在蛋白质 3-D 支架上的空间分布很广泛，分为两种我们称之为“金属结合区”（MBR）和“野生型样”（WTL）突变体的类别[10]。我们之前的对 SOD1 MBR 突变体类的五个成员的晶体学研究表明它们是金属缺乏。金属离子的缺失会导致环元件发生构象变化，从而使边缘脱保护蛋白质中的β-折叠链。这种保护的丧失反过来会导致“互动增益”（GOI）突变型 SOD1 二聚体之间的相互作用可促进线性和螺旋丝状阵列的形成 [1]。因此，金属缺乏酶中的构象重排导致更高阶的寡聚体组装可以代表与 FALS 相关的 SOD1 突变体共有的毒性特性。 SOD1 的 WTL 突变体类别的一些成员的脱辅基蛋白形式非常不稳定，以至于它们可能在它们有机会被适当地元化或二聚化之前聚集[11]。然而，有其他 WTL 突变体在金属化和脱辅基形式方面与 WT SOD1 一样稳定（参见初步结果，项目 1)。对这一明显矛盾的一个可能的解释是，后者 WTL 突变体 SOD1 蛋白由于与异常相互作用而在体内未正确金属化金属伴侣。另一种可能性是这些突变体在体内更容易被氧化损伤，氧化的致病蛋白的金属结合能力受损，因此更容易容易发生错误折叠和/或聚集。该研究项目（项目 2）的目标是更好地了解通过使用多种互补的生物物理方法聚集致病性 SOD1 将这些信息与我们共同努力探索的决定因素所获得的信息结合起来这些蛋白质在体内聚集。对致病性 SOD1 聚集的基本理解是未来努力获得针对蛋白质的疾病的治疗剂的先决条件错误折叠和/或蛋白质降解途径。