Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species
Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species
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DOI：
10.1016/j.jmb.2010.02.034
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发表时间：
2010-04-30
影响因子：
5.6
通讯作者：
Matthews, C. Robert
中科院分区：
文献类型：
--
作者：
Kayatekin, Can;Zitzewitz, Jill A.;Matthews, C. Robert
铜锌超氧化物歧化酶（SOD 1）是一种二聚体金属结合酶，负责在细胞内将有毒的超氧化物歧化为过氧化氢和氧气。SOD1中数十个位点的突变诱导肌萎缩侧索硬化症（ALS），这是一种致命的功能获得性神经退行性疾病，其分子基础尚不清楚。为了深入了解突变对未成熟单体形式的折叠和未折叠群体的影响，这些未成熟单体形式的聚集或自缔合可能是ALS的原因，将一组二硫键还原和二硫键氧化的无Zn和Zn结合的稳定单体SOD 1变体的热力学和动力学折叠性质与野生型（WT）蛋白质的性质进行比较。对于二硫化物还原的无金属变体，观察到突变对单体稳定性的最显著影响。WT和S134N单体在中性和37 ° C下折叠>95%，而A4V、L38V、G93A和L106V在50%至类似于90%的范围内未折叠。还发现二硫键的还原将WT单体的表观Zn亲和力降低750倍，进入纳摩尔范围，其中其可能无法竞争细胞中的游离Zn。除了S134 N金属结合变体外，二硫键氧化的SOD 1单体的Zn亲和力对氨基酸置换几乎不敏感。这些结果表明了一个模型的SOD1聚集的核糖体上的不断合成的ALS变体的SOD1提供了一个池的物种，其中人口增加的未折叠状态可能有利于聚集生产折叠到天然二聚体状态。(C)2010爱思唯尔有限公司保留所有权利。
Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wildtype (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to similar to 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state. (C) 2010 Elsevier Ltd. All rights reserved.