Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals
Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals
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DOI：
10.1016/j.aquatox.2012.02.007
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发表时间：
2012-06-15
影响因子：
4.5
通讯作者：
Santovito, G.
中科院分区：
文献类型：
--
作者：
Franchi, N.;Ferro, D.;Santovito, G.
已知暴露于金属会在生物体中产生氧化应激风险，生物体能够通过诱导酶促和非酶促的抗氧化防御反应。谷胱甘肽（GSH）被认为是一种重要的细胞成分，参与保护细胞，无论是作为金属螯合剂和氧自由基清除剂。本研究利用分子生物学技术分析了单囊玻璃海鞘中GSH合成相关基因γ-谷氨酰-半胱氨酸连接酶催化亚基（ci-gclc）、γ-谷氨酰-半胱氨酸连接酶修饰亚基（ci-gclm）和GSH合成酶（ci-gs）的核苷酸序列和预测的氨基酸序列。为了进一步了解金属诱导的氧化应激与GSH生成的关系，我们还采用半定量RT-PCR技术研究了Cd、Cu和Zn在体内暴露后上述基因的转录情况，并采用原位杂交技术对mRNA表达进行了定位。这些基因通常与后生动物同源物表现出良好的序列保守性，特别是对于酶活性重要的残基。系统发育分析表明，这三种酶以不同的方式进化。Ci-GCLC和Ci-GS主要与无脊椎动物蛋白质相关，Ci-GCLM是脊椎动物GCLM的姐妹群。我们的计算机模拟分析的ci-gs和ci-gclc启动子区域揭示了推定的共识序列类似于哺乳动物金属响应元件（MRE）和抗氧化剂响应元件（ARE），表明这些基因的转录可能直接依赖于金属和/或活性氧。结果突出了基因转录的统计学显著增加，表明金属处理对这些基因具有诱导作用。它们不仅可以通过MRE调节基因转录，还可以通过战神调节基因转录，这是金属依赖性ROS形成的结果。Ci-GS和Ci-GCLC mRNA的ISH定位显示，参与谷胱甘肽生物合成最多的细胞是循环血细胞。本文提供的数据强调了复合金属调控ci-gclc、ci-gclm和ci-gs转录的重要性，这可以创建一个有效的解毒途径，使C.在环境中金属含量持续升高的情况下，它们难以生存。(c)2012 Elsevier B. V.保留所有权利。
Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, gamma-glutamyl-cysteine ligase catalytic subunit (ci-gclc), gamma-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways. Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment. (c) 2012 Elsevier B.V. All rights reserved.