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Structure and Function of Mutant SOD1 Derived from 2 bp Deletion in the Gene

基因中 2 bp 缺失衍生的突变体 SOD1 的结构和功能

基本信息

批准号：
09680621
负责人：
SHIBATA Hitoshi
金额：
$ 2.11万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680621/
关键词：
SOD FALS fused-protein 筋萎縮性側索硬化症 2塩基欠損遺伝子異常異常タンパク

项目摘要

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons of cortex, brainstem, and spinal cord. About 10% of all ALS are familial cases, which are inherited as an autosomal dominant trait. Recent studies showed that about 20% of familial ALS (FALS) cases have mutations in SOD1, the gene for Cu, Zn-superoxide dismutase (SOD) that catalyzes the dismutation of superoxide radical anions to hydrogen peroxide and oxygen molecules. All of the FALS mutations do not change any active site residues involving the coordination of the metal ions or residues forming the electrostatic active channel. Rather, most of the FALS mutant sites alter conserved interactions critical to the subunit fold and dimer contact.The mutant SOD associated with FALS found in Sanin area north western part of Japan was derived from 2 bp deletion in the SOD1. The SOD activity in extracts from red blood cells, brain tissues, and lymphoblastoid cells revealed an about 50% reduction in FALS patients co … More mpared to normal individuals. The human SOD1 was expressed as a fusion protein in Eschericia coli, then purified by an affinity chromatography. The SOD activity and protein band clearly demonstrated even in the fused states. But, the fused protein expressed from the mutant SOD1 showed clear but very low activity, After the separation of mutant SOD from the fused protein by Factor Xa, the activity and the protein band on the gel disappeared during the purification process. As the 2 bp deletion was found to be very important, by analyzing the three dimensional modeL structure, to compose the dimeric active structure, The dimer structure of the mutant SOD is very labile, thus copper ions released from the SOD molecules will be the source of oxidative stress in tissues of patients. Although the association mechanism of the mutations of SOD with FALS is still unknown, the lowered SOD activity may not be associated FALS but the SOD mutants retain high levels of peroxidative activity (the gain-of-function), and utilize its own dismutation product producing the main species hydroxyl radicals which is the source of oxidative stress. Less

肌萎缩侧索硬化症（ALS）是一种皮质、脑干和脊髓运动神经元的退行性疾病。大约 10% 的 ALS 是家族性病例，作为常染色体显性遗传。最近的研究表明，大约 20% 的家族性 ALS (FALS) 病例存在 SOD1 突变，SOD1 是铜、锌超氧化物歧化酶 (SOD) 的基因，可催化超氧自由基阴离子歧化为过氧化氢和氧分子。所有 FALS 突变不会改变任何涉及金属离子配位的活性位点残基或形成静电活性通道的残基。相反，大多数 FALS 突变位点改变了对亚基折叠和二聚体接触至关重要的保守相互作用。在日本西北部山阴地区发现的与 FALS 相关的突变 SOD 源自 SOD1 中的 2 bp 缺失。红细胞、脑组织和类淋巴母细胞提取物中的 SOD 活性显示，与正常人相比，FALS 患者的 SOD 活性降低了约 50%。人SOD1在大肠杆菌中表达为融合蛋白，然后通过亲和层析纯化。即使在融合状态下，SOD 活性和蛋白质条带也清晰可见。但是，突变体SOD1表达的融合蛋白显示出清晰但非常低的活性，在通过因子Xa将突变体SOD与融合蛋白分离后，在纯化过程中活性和凝胶上的蛋白条带消失。由于发现2bp缺失非常重要，通过分析三维modelL结构，组成二聚体活性结构，突变体SOD的二聚体结构非常不稳定，因此SOD分子释放的铜离子将成为患者组织氧化应激的来源。尽管SOD突变与FALS的关联机制尚不清楚，但SOD活性降低可能与FALS无关，但SOD突变体保留了高水平的过氧化活性（功能获得性），并利用其自身的歧化产物产生主要种类羟基自由基，这是氧化应激的来源。较少的