Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns
Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns
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DOI：
10.1016/j.bbagen.2012.01.012
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发表时间：
2012-04-01
影响因子：
3
通讯作者：
De Giorgi, Carla
中科院分区：
文献类型：
--
作者：
Liuzzi, Vania C.;Giancaspero, Teresa A.;De Giorgi, Carla
背景：FAD合成酶是一种普遍存在的酶，它催化FAD生物合成的最后一步，允许几种黄素蛋白的生物发生。在人类中，不同的异构体通过选择性剪接产生，异构体1定位于线粒体。在秀丽隐杆线虫中进行同源性搜索，鉴定出两个人类FAD合成酶同源物，由单拷贝基因R53.1编码。方法：采用饲养法沉默秀丽隐杆线虫R53.1基因。采用半定量RT-PCR法测定转录本的表达水平。用双向电泳法测定总蛋白组成。用分光光度法和极谱法测定分离线粒体的酶活性和耗氧量。结果：R53.1通过反式剪接产生两个转录本。RNAi使R53.1的转录效率降低50%，导致总黄酮减少50%，ATP含量降低，ROS水平升高。在敲除线虫中发现了显著的表型变化。其中，运动行为的显著损伤可能是由于胆碱能传递的改变。在生化水平上，检测到黄酶活性和一些kcn不敏感的耗氧酶的损伤。在蛋白质组学水平上，至少有15种丰富的蛋白质受到R53.1基因沉默的影响，其中包括超氧化物歧化酶。结论和一般意义：我们首次发现线虫中存在不同的fad代谢酶同工型。FAD合酶沉默与黄酮类酶紊乱、能量短缺和氧化还原平衡损害之间存在明显的相关性。在这方面，r53.1干扰线虫可以为研究黄素稳态/黄酶生物发生改变的人类病理提供动物模型系统。(C) 2012 Elsevier B.V.版权所有
Background: FAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1.Methods: The C elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria.Results: From R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases.Conclusion and General Significance: For the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis. (C) 2012 Elsevier B.V. All rights reserved.