Regulation of the Dynamic Proteome after Ischemic Injury

摘要 这项工作的前提是基于积累的证据，即线粒体重塑发生在细胞内。 缺血性损伤后的应激心脏，不仅发生在危险区域，而且还发生在剩余的存活区域。 心肌现在必须改变其工作能力。我们之前发现缺血和再灌注 导致在RNA翻译水平上调节线粒体自噬和生物发生。本应用程序将探讨这一点 通过检查心脏中活性多聚核糖体的动态蛋白质组成， 缺血性应激目的1将检验调节分子与多聚核糖体结合的假说， 缺血性应激，并在这样做时选择靶向mRNA进行主动翻译。将进一步 确定miRNAs，或者确切地说是某些miRNAs的丢失，是否也控制了这一过程。本项目的成果 将开发一个新的合成的蛋白质在缺血恢复设置的详细画像，使用 用质谱兼容的Met类似物（叠氮基高丙氨酸，AHA）进行代谢标记，以确定新的 蛋白质组目标2中的工作将定义线粒体功能改变与 改变蛋白质乙酰化和转录输出的代谢组学变化。在这方面的工作将雇用 干扰糖酵解和氧化磷酸化的各种干预，表征代谢组， 将结果与蛋白质乙酰化和转录组的特定改变相关联，包括mRNA和 小RNA。还将进行实验，以探索扰动，包括高脂肪饮食和变化， 线粒体单倍型群[使用胞质杂种细胞和线粒体核交换（MNX）小鼠系]。的 多组学分析将需要开发新的生物信息学工具，以确定关系和控制 节点通过代谢物水平的变化介导。我们已经确定了一条共同的道路， 通过吡格列酮和GLP 1 Ra增加NADH/NAD+比率，抑制sirtuins，导致蛋白质 乙酰化，并降低miR-33的表达和活性。这些研究将揭示 线粒体生物合成的翻译控制，这可能导致新的治疗工具的发展， 在缺血/再灌注损伤的情况下调节该过程。第二和第三个目标将产生一个新的 了解代谢和线粒体输出对转录的作用，预计 鉴定在转录调节中特别重要的代谢物，其可能支配心脏重塑。 该项目的重点是梗死后重塑的机制，并将产生一个新的理解， 存活心肌中线粒体功能、代谢输出和转录调控之间的联系 梗塞后。

ABSTRACT The premise of this work is based on the accumulated evidence that mitochondrial remodeling takes place in the stressed heart after ischemic injury, and occurs not only in the area at risk, but also in the remaining viable myocardium that must now alter its work capacity. We previously discovered that ischemia and reperfusion resulted in mitophagy and biogenesis regulated at the level of RNA translation. This application will explore this finding further by examining the dynamic protein composition of the active polysomes in hearts subjected to ischemic stress. Aim 1 will test the hypothesis that regulatory molecules associate with polysomes during ischemic stress and, in doing so, select mitochondria-targeted mRNAs for active translation. It will further determine if miRNAs, or rather the loss of certain miRNAs, also govern this process. Results from this project will develop a detailed portrait of the newly-synthesized proteins in the setting of recovery from ischemia, using metabolic labeling with a mass spectrometry-compatible Met analog (azidohomoalanine, AHA) to define the new protein proteome. Work in Aim 2 will define the relationship between alterations in mitochondrial function and changes in the metabolome that alter protein acetylation and transcriptional output. Work in this aim will employ various interventions to perturb glycolysis and oxidative phosphorylation, characterize the metabolome, and correlate results with specific alterations in protein acetylation and the transcriptome, including both mRNA and miRNA. Experiments will also be performed to explore perturbations, including a high-fat diet and variations in mitochondrial haplogroup [using cybrid cells and the mitochondrial nuclear exchange (MNX) mouse lines]. The multi-omics analysis will require the development of novel bioinformatics tools to identify relationships and control nodes mediated through changes in metabolite levels. We have already identified a common pathway shared by pioglitazone and GLP1Ra which increase the NADH/NAD+ ratio, inactivate sirtuins, result in increased protein acetylation, and lower the expression and activity of miR-33. These studies will reveal the mechanisms governing translational control of mitochondrial biogenesis, which may lead to the development of new therapeutic tools for regulating this process in the setting of ischemia/reperfusion injury. The second and third aims will yield a new understanding of the role that metabolism and mitochondrial output has on transcription, and is expected to identify metabolites of particular importance in transcriptional regulation that may govern cardiac remodeling. This project is focused on mechanisms of post-infarction remodeling and will yield a new understanding of the link between mitochondrial function, metabolic output, and transcriptional regulation in the surviving myocardium after infarction.