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.

Sod 1成熟过程的分子机制 铜离子受到需氧生物的严格调节，以至于不稳定的铜几乎检测不到。 虽然浓度升高是有毒的，但氧化态之间相互转化的能力（例如Cu（I）和Cu（II）） 使得铜离子可用于多种细胞过程。铜（和其他氧化还原活性金属离子） 通过一组不同的螯合剂， 包括运输蛋白和分子清除剂。金属伴侣是一个多样化的家庭贩运 为蛋白质靶提供金属离子以用作辅因子的分子。其中一个目标是无处不在的 抗氧化酶铜锌超氧化物歧化酶（SOD 1）。Sod 1的铜分子伴侣（Ccs 1） 将单个铜离子传递到活性位点，并催化Sod 1内二硫键的氧化， 一个机械上模棱两可的过程。这些Ccs 1介导的翻译后修饰转化 将不成熟的Sod 1从一系列边缘稳定的无活性单体转化为一种非常稳定的活性单体， 同二聚体。编码Sod 1的基因突变与致命的神经退行性疾病有关。 肌萎缩侧索硬化症（ALS）。有趣的是，正是这些致病性Sod 1的不成熟形式 在易感细胞中发现的构成疾病相关聚集体的变异。吾等已厘定 全长Ccs 1的结构在2.35 ℃下与未成熟形式的Sod 1结合。杂合物的结构 揭示了先前未观察到的Ccs 1 C-末端结构域（D3）的β-发夹构象，表明 Ccs 1动作的“枢轴和释放”机制。我们认为Ccs 1与未成熟形式的SOD 1结合， 诱导Sod 1中保守环元件的运动，暴露出正电性孔，并提出 铜离子传递到Sod 1的“进入位点”。由于我们目前的Sod 1·Ccs 1杂合物结构， 使用以下方法研究Ccs 1作用的基于结构的方法（I）基于荧光的方法 系统设计来描绘热力学引导Ccs 1介导的野生型和突变体的成熟 Sod 1的形式，（II）在渐进步骤中额外的Sod 1·Ccs 1杂合物的结构表征， 成熟过程，（III）X射线吸收光谱及相关技术探测铜离子 协调在Sod 1“进入网站”和（IV）基于细胞的生化分析，重点是连接催化 在Sod 1“进入位点”的周转和二硫键形成和释放的亚磺酸中间体 铜离子到Sod 1活性位点。这里提出的目标旨在结束正在进行的辩论 关于Sod 1通过Ccs 1成熟的普遍机制，可能有助于说明 Sod 1相关的神经退行性疾病ALS。