Oxidative stress in Caenorhabditis elegans:: protective effects of the Omega class glutathione transferase (GSTO-1)
Oxidative stress in Caenorhabditis elegans:: protective effects of the Omega class glutathione transferase (GSTO-1)
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DOI：
10.1096/fj.06-7426com
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发表时间：
2008-02-01
期刊：
影响因子：
4.8
通讯作者：
Liebau, Eva
中科院分区：
文献类型：
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作者：
Burmeister, Cora;Lueersen, Kai;Liebau, Eva
为了阐明Omega类谷胱甘肽转移酶（GSTs）（EC 2.5.1.18）在多细胞生物中的功能，研究了来自秀丽隐杆线虫的GSTO-1（GSTO-1; C29E4.7）。使用过表达GSTO-1的大肠杆菌的圆盘扩散测定提供了对氧化应激下长期暴露的抗性的测试。亲和纯化后，重组GSTO-1对经典GST底物具有最小的催化活性，但显示出显着的巯基氧化还原酶和脱氢抗坏血酸还原酶活性。显微注射GSTO-1启动子绿色荧光蛋白构建体和免疫定位的电子显微镜定位的蛋白质只在肠道的所有胚后阶段的C。优雅缺失分析鉴定了GSTO-1表达所必需的ATG起始位点上游的类似于300个核苷酸的序列。最小启动子中的加塔转录因子结合基序的位点特异性诱变导致报告基因表达的丧失。类似地，Elt-2的RNA干扰（RNAi）表明该肠道特异性转录因子参与GSTO-1表达。通过分析转基因C. elegans菌株。为了研究GSTO-1在体内的功能，产生过表达GSTO-1的转基因动物，其表现出对胡桃醌、百草枯和枯烯过氧化氢诱导的氧化应激的抗性增加。通过RNAi特异性沉默GSTO-1产生的蠕虫对几种促氧化剂、亚砷酸盐和热休克的敏感性增加。我们的结论是应激反应GSTO-1在对抗环境胁迫中起着关键作用。
To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO-1 from Caenorhabditis elegans (GSTO-1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress. After affinity purification, the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an similar to 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional upregulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.