Effects of environmental stressors on mitochondrial-cellular cross-talk

 描述(由申请人提供)：这份R21拨款申请拨款申请符合研究目标中所述的目标。具体地说，其目的是在果蝇组织中建立一个强大的平台，以表征线粒体(活性、蛋白质组成)和基因组(核编码的线粒体基因和其他)在暴露于环境线粒体应激源(如有毒金属)时发生的变化。最初的研究将集中在肠道上，因为它是金属首先被吸收的组织。这些研究的结果将使人们能够设计携带环境线粒体毒物生物标记物的苍蝇，这些生物标记物可用于剖析线粒体和细胞核之间串扰和信号传递的分子机制。赠款申请由三个特定目标组成：特定目标1)表征成年果蝇肠道中线粒体活性和蛋白质组组成对环境线粒体毒物的响应变化。线粒体活性，包括柠檬酸合成酶活性和耗氧率，以及线粒体蛋白质组组成，将在有毒金属喂养后的不同时间点进行测量。在蛋白质组学研究方面，将开发一种新的基于工程抗坏血酸过氧化物酶(APEX)的蛋白质标记技术，该技术最近被用于果蝇活组织的研究；具体目的2)表征果蝇肠道对环境线粒体毒物的转录变化。将在不同的时间点检测肠道的RNA-Seq，以表征在喂养有毒金属后该组织中发生的转录变化。此外，使用最近开发的Compleat工具，将确定优先受环境线粒体毒物影响的线粒体蛋白质复合体；并专门针对3)产生环境线粒体应激的生物标记物。将选择由环境线粒体应激源诱导的基因，使用CRISPR、GFP传感器进行工程设计，这种传感器可用于剖析线粒体毒物反应。在R21阶段之后，我们设想将这个项目扩展到R33，我们将：1)研究额外的环境线粒体应激源，以建立特定毒物的特征；2)扩展到其他组织(脑、肌肉和脂肪组织)，以获得对不同类型细胞反应的多样性的一般了解；以及 3)使用携带环境线粒体毒物生物标记物的工程苍蝇来剖析线粒体和细胞核之间串扰和信号传递的分子机制。

 DESCRIPTION (provided by applicant): This R21 grant application grant application addresses the goals stated in the Research Objectives. Specifically, the intent is to establish a robust platform in Drosophila tissues to characterize changes occurring in mitochondria (activity, protein composition), and in the genome (nuclear-encoded mitochondrial genes and others), in response to exposure to environmental mitochondrial stressors, such as toxic metals. The initial studies will focus on the gut, as it is the tissue where metals are first absorbed. Results from these studies will allow one to engineer flies carrying biomarkers of environmental mitochondrial toxicants that can be used to dissect the molecular mechanisms underlying cross-talk and signaling between mitochondria and the nucleus. The grant application is composed of three specific aims: Specific Aim 1) Characterize changes in mitochondrial activities and proteome constitution in response to environmental mitochondrial toxicants in the adult Drosophila gut. Mitochondrial activities, including citrate synthase activity and O2 consumption rate, as well as mitochondrial proteome constitution, will be measured at different timepoints following toxic metals feedings. For the proteomic studies, a novel protein labeling technique will be exploited, based on an engineered ascorbate peroxidase (APEX), that was recently adapted for studies in Drosophila live tissues; Specific Aim 2) Characterize the transcriptional changes occurring in the Drosophila gut in response to environmental mitochondrial toxicants. RNA-Seq of gut will be examined at different timepoints to characterize the transcriptional changes occurring in this tissue following feeding of toxic metals. Further, using the recently developed COMPLEAT tool, mitochondrial protein complexes that are preferentially affected by environmental mitochondrial toxicants will be determined; and Specific Aim 3) Generate biomarkers of environmental mitochondrial stress. Genes will be selected that are induced by environmental mitochondrial stressors to engineer, using CRISPR, GFP sensors that can be used to dissect the mitochondrial toxicant response. Following the R21 phase, we envision expanding this project into an R33 where we will: 1) Study additional environmental mitochondrial stressors to establish signatures of specific toxicants; 2) Expand to other tissues (brain, muscle, and adipose tissues) to gain a general understanding of the diversity of responses of different cell types; and 3) Use engineered flies carrying biomarkers of environmental mitochondrial toxicants to dissect the molecular mechanisms underlying cross-talk and signaling between mitochondria and the nucleus.