The structural analysis of the pro-oxidant copper-binding site of denatured apo-H43R SOD1 and the elucidation of the origin of the acquisition of the pro-oxidant activity
The structural analysis of the pro-oxidant copper-binding site of denatured apo-H43R SOD1 and the elucidation of the origin of the acquisition of the pro-oxidant activity
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DOI：
10.1039/c5cp07729j
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发表时间：
2016-02-14
影响因子：
3.3
通讯作者：
Nakabayashi, Takakazu
中科院分区：
文献类型：
--
作者：
Fujimaki, Nobuhiro;Miura, Takashi;Nakabayashi, Takakazu
肌萎缩性侧索硬化症（ALS）的发病机制与Cu， zn -超氧化物歧化酶（SOD1）突变有关，SOD1是一种具有代表性的抗氧化酶。先前的研究表明，人类SOD1与ALS相关的突变体His43 -> Arg （H43R）的变性载脂蛋白形式通过重新结合Cu2+获得了与天然抗氧化活性相反的促氧化活性，这被认为与ALS的发展密切相关。变性apo-H43R中的Cu2+结合位点可视为促氧化活性的中心，引起细胞氧化应激。本研究对变性apo-H43R的Cu2+结合位点的结构进行了研究，以阐明其促氧化活性的获得机制。通过SOD1突变体的吸收和荧光分析，实验确定了变性apo-H43R中构建Cu2+结合位点的His残基，其中H43R SOD1中的7个His残基都被Ala取代。发现在变性后，His120不参与Cu2+结合位点，尽管其他构建金属结合位点的His残基在变性后保持不变。因此，从Cu2+结合位点上消失的His120被认为是获得促氧化活性的重要因素之一。在此基础上，讨论了促氧化活性的获得机理。
The pathogenesis of amyotrophic lateral sclerosis (ALS) is associated with mutations of Cu,Zn-superoxide dismutase (SOD1), which is a representative antioxidant enzyme. A previous study showed that the denatured apo-form of an ALS-linked mutant of human SOD1, His43 -> Arg (H43R), obtains pro-oxidant activity as the reverse behavior of the native antioxidant activity by rebinding Cu2+, which is considered to be closely related to the development of ALS. The Cu2+-binding site in denatured apo-H43R can be regarded as the center of the pro-oxidant activity, causing cellular oxidative stress. In the present study, the structure of the Cu2+-binding site of denatured apo-H43R was investigated to clarify the mechanism of the acquisition of the pro-oxidant activity. His residues constructing the Cu2+-binding site in denatured apo-H43R were experimentally assigned by absorption and fluorescence-based assays of SOD1 mutants, in which each of the seven His residues in H43R SOD1 is replaced with Ala. It was found that His120 is not involved with the Cu2+-binding site after denaturation, although the other His residues constructing the metal-binding site remain constant after denaturation. The disappearance of His120 from the Cu2+-binding site is therefore considered to be one of the important factors in obtaining the pro-oxidant activity. The mechanism of the acquisition of the pro-oxidant activity is discussed based on the results obtained.