Molecular Mechanisms of Sod1 Maturation Processes

Sod1 成熟过程的分子机制

• 批准号: 9489285
• 负责人: Duane David Winkler
• 金额: $ 27.44万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9489285
• 关键词: Active Sites; Aerobic; Affinity; Amyotrophic Lateral Sclerosis; Binding; Biochemical; C-terminal; Cell physiology; Cells; Code; Coenzymes; Collection; Complex; Copper; Cuprozinc Superoxide Dismutase; Cysteine; Cytosol; Dioxygen; Disease; Disulfides; Elements; Ensure; Event; Exhibits; Family; Fluorescence; Genes; Goals; Homodimerization; Ions; Knock-out; Length; Link; Mammalian Cell; Mediating; Metalloproteins; Metals; Methods; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Mechanisms of Action; Movement; Mutation; N-terminal; Neurodegenerative Disorders; Organism; Outcome; Oxidation-Reduction; Oxidative Stress; Oxygen; Pathogenicity; Pathway interactions; Phenotype; Play; Post-Translational Protein Processing; Process; Property; Proteins; Reaction; Research; Roentgen Rays; Role; Site; Spectrum Analysis; Structure; Suggestion; Sulfenic Acids; Superoxide Dismutase; Superoxides; System; TRAP Complex; Techniques; Thermodynamics; Translating; Tryptophan; Variant; Work; X-Ray Crystallography; Yeasts; Zinc; absorption; antioxidant enzyme; base; cofactor; copper zinc superoxide dismutase; design; disulfide bond; monomer; mutant; oxidation; physical property; prevent; protein transport; superoxide dismutase 1; tool; trafficking; uptake; virtual

Molecular Mechanisms of Sod1 Maturation Processes Copper ions are tightly regulated by aerobic organisms to the extent that labile copper is virtually undetectable. Although elevated levels are toxic, the capacity to interconvert between oxidation states (e.g. Cu(I) and Cu(II)) makes copper ions useful for a wide variety of cellular processes. Copper (and other redox-active metal ions) is constantly guarded from promiscuous reactions in the cytosol by a diverse group of sequestering agents that include trafficking proteins and molecular scavengers. Metallo-chaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. One such target is the ubiquitous antioxidant enzyme copper-zinc superoxide dismutase (Sod1). The copper chaperone for Sod1 (Ccs1) delivers a single copper ion to the active-site and catalyzes oxidation of the disulfide bond within Sod1 through a mechanistically ambiguous process. These Ccs1-mediated posttranslational modifications transform immature Sod1 from a collection of marginally stable, inactive monomers into a remarkably stable, active homodimer. Mutations in the gene that code for Sod1 have been implicated in the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Interestingly, it is the immature forms of these pathogenic Sod1 variants that make up the disease related aggregates found in susceptible cells. We have determined the structure of full-length Ccs1 bound to an immature form of Sod1 at 2.35 Å. The structure of the heterocomplex reveals a previously unobserved β-hairpin conformation of the Ccs1 C-terminal domain (D3) suggestive of a “pivot and release” mechanism for Ccs1 action. We suggest that Ccs1 binding to an immature form of Sod1 induces movement of a conserved loop element in Sod1 that exposes an electropositive hole and proposed “entry site” for copper ion delivery to Sod1. We have since our current Sod1•Ccs1 heterocomplex structure for a structure-based approach to investigate Ccs1 action using the following methods (I) a fluorescence based system designed to delineate the thermodynamics guiding Ccs1-mediated maturation of wild-type and mutant forms of Sod1, (II) structural characterization of additional Sod1•Ccs1 heterocomplexes at progressive steps in the maturation process, (III) X-ray absorption spectroscopy and related techniques to probe copper ion coordination at the Sod1 “entry site” and (IV) cell-based biochemical analysis focusing on connecting catalytic turnover at the Sod1 “entry site” and a sulfenic acid intermediate to disulfide bond formation and release of the copper ion to the Sod1 active site. The aims presented here are designed to end the ongoing debate regarding a universal mechanism for Sod1 maturation via Ccs1 and may help to illustrate the initial stages of the Sod1-linked neurodegenerative disorder ALS.

SOD1成熟过程的分子机制 铜离子受氧生物的严格调节，以至于虚弱的铜几乎无法检测到。 尽管水平升高是有毒的，但在氧化态之间相互转化的能力（例如Cu（i）和Cu（ii）） 铜离子可用于多种细胞过程。铜（以及其他氧化还原活性金属离子） 不断受到一群隔离药物的群体的滥交反应，这些反应是 包括贩运蛋白和分子清除剂。金属座是一个多元化的贩运家族 为蛋白质靶标提供金属离子以用作辅助因子的分子。一个这样的目标是无处不在 抗氧化剂铜锌超氧化物歧化酶（SOD1）。 SOD1的铜伴侣（CCS1） 将单个铜离子传递到主动位置，并通过SOD1中的二硫键氧化通过 机械模棱两可的过程。这些CCS1介导的翻译后修饰变换 来自边缘稳定的，不活跃的单体的未成熟SOD1到一个非常稳定的活性 同二聚体。在致命神经退行性中暗示了基因中的突变 肌营养不良的侧向硬化症（ALS）。有趣的是，这是这些致病性SOD1的未成熟形式 构成易感细胞中与疾病相关的聚集体的变体。我们已经确定了 全长CCS1的结构与2.35Å时SOD1的未成熟形式结合。杂点的结构 揭示了CCS1 C末端结构域（D3）的先前未观察到的β-发率构象 CCS1动作的“枢轴和释放”机制。我们建议CCS1与SOD1的未成熟形式结合 在SOD1中诱导保守环元件的运动，该元件暴露了电孔并提议 将铜离子传递到SOD1的“入口站点”。自我们当前的SOD1•CCS1杂音结构以来 一种基于结构的方法来研究CCS1动作，使用以下方法（i）基于荧光的方法 旨在描述热力学指导CCS1介导的野生型和突变体的成熟的系统 SOD1的形式，（ii）附加SOD1的结构表征•CCS1杂音在渐进的步骤中 成熟过程，（iii）X射线抽象光谱和探测铜离子的相关技术 SOD1“入口站点”和（IV）基于细胞的生化分析的协调，重点是连接催化 SOD1“入口部位”和硫酸中间体的营业额，以二硫键形成和释放 铜离子到SOD1活性位点。这里提出的目的旨在结束正在进行的辩论 考虑通过CCS1成熟SOD1成熟的通用机制，可能有助于说明 SOD1连接的神经退行性疾病ALS。