Molecular Mechanisms of Sod1 Maturation Processes

SOD1成熟过程的分子机制 铜离子受到好氧生物的严格控制，以至于几乎检测不到不稳定的铜。 虽然水平升高是有毒的，但在氧化态(如铜(I)和铜(II))之间相互转换的能力 使铜离子在多种细胞过程中发挥作用。铜(和其他氧化还原活性金属离子) 由一组不同的隔离剂持续保护，防止细胞质中的混杂反应 包括运输蛋白质和分子清道夫。金属伴侣是一个多样化的人口贩运家族 为蛋白质靶标提供金属离子以用作辅因子的分子。一个这样的目标是无处不在的 抗氧化酶铜锌超氧化物歧化酶(SOD1)。SOD1(Ccs1)的铜伴侣 将单个铜离子传递到活性中心，并通过以下方式催化SOD1中二硫键的氧化 机械上模棱两可的过程。这些CCS1介导的翻译后修饰将 不成熟的SOD1从边缘稳定、不活跃的单体转变为非常稳定、活跃的 同源二聚体。编码SOD1的基因突变与致命的神经退行性变有关 肌萎缩侧索硬化症(ALS)。有趣的是，正是这些致病的SOD1的不成熟形式 在敏感细胞中发现的构成疾病相关聚集体的变体。我们已经确定了 全长Ccs1的结构与未成熟形式的SOD1结合在2.35？杂化络合物的结构 揭示了以前未观察到的CCS1 C-末端结构域(D3)的β-发夹构象，提示 CCS1操作的“枢轴和释放”机制。我们认为Ccs1与一种不成熟的SOD1结合 在SOD1中诱导保守环状元件的移动，暴露出一个正电孔，并提出 铜离子向SOD输送的“入口点”。自从我们目前的SOD1.Ccs1杂络结构以来，我们已经 使用以下方法研究CCS1作用的基于结构的方法(I)基于荧光的 用于描述Ccs1介导的野生型和突变体成熟的热力学系统 SOD_1的形式，(II)额外的SOD_1·CCS_1杂化配合物的结构表征 成熟过程、(III)X射线吸收光谱及相关的铜离子探测技术 SOD1“入口点”的协调和(IV)以细胞为基础的生化分析，重点是连接催化剂 SOD1“进入位”的周转率和磺酸中间体二硫键的形成和释放 铜离子对SOD1活性中心的影响。这里提出的目的是为了结束正在进行的辩论 关于SOD1通过CCS1成熟的普遍机制，并可能有助于说明 SOD1相关的神经退行性疾病ALS。

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.