Translational mechanisms of mitochondrial protein synthesis

描述(申请人提供)：尚不清楚哺乳动物细胞是否或如何诱导线粒体蛋白质的选择性翻译，以及这是否因细胞/组织类型而不同。这项研究的长期TRM目标是确定可用于增加人类线粒体蛋白质合成的策略，以增加健康寿命。总体目标是确定在哺乳动物细胞/组织能量应激期间增加线粒体蛋白质合成的转录后机制。中心假设是，在能量应激期间观察到的哺乳动物肝脏、骨骼肌和心脏中线粒体蛋白的选择性翻译是由mRNAs序列元件的复杂性和/或线粒体网状结构的亚细胞定位决定的。这一假设是基于申请者实验室中产生的证据以及在低等真核生物中广泛发表的证据而提出的。这项拟议研究的基本原理是，这项研究的成功完成将提供新的机会，通过靶向几乎没有在哺乳动物细胞中研究的机制来确定延缓衰老和延长健康寿命的治疗靶点。这项研究有两个特定的目的：1)在体外确定哺乳动物线粒体蛋白质在能量应激信号中优先翻译和合成的机制，以及自噬/有丝分裂吞噬对这些增加的贡献程度；2)确定激活体内能量应激途径以保护线粒体蛋白质合成和延缓衰老的策略。为了实现这些特定的目标，将分析多种细胞类型和组织。体外使用的方法包括快速翻译mRNA的多聚体图谱分析，mRNA序列的生物信息学分析，以及用于测定线粒体蛋白质合成和自噬通量的新的稳定同位素技术。体内使用的方法包括其他新的同位素技术，用于测量组织、细胞器和个体蛋白质水平的蛋白质合成以及细胞增殖。这项拟议的研究的贡献是重大的，因为它旨在阐明线粒体生物发生的一个重要但未被研究的调节步骤。此外，它的设计是将体外机制转化为体内研究，以确定这些机制是否可以作为治疗的靶点。这项拟议的研究具有创新性，因为它考虑了线粒体生物发生的转录后机制，这些机制以前在很大程度上被忽视了。此外，该实验室是少数几个能够进行长期蛋白质合成和细胞增殖测量的小组之一，这些测量允许将体外研究转化为体内结果。最后，到目前为止，对单个线粒体蛋白质合成的体内评估还不可行，因此该项目具有技术创新。预计这些结果将产生积极影响，因为它们侧重于可能提供新的治疗靶点的机制，这些机制可以替代一种治疗方法(终生热量限制)，这种疗法虽然有效，但在人类身上的依从性极其有限。

DESCRIPTION (provided by applicant): It is not known if or how mammalian cells induce selective translation of mitochondrial proteins and if this differs by cell/tissue type. The long-trm goal of this research is to identify strategies that can be used to increase mitochondrial protein synthesis in humans for the purpose of increasing healthspan. The overall objective is to identify post-transcriptional mechanisms that are involved in increasing mitochondrial protein synthesis during periods of energetic stress in mammalian cells/tissues. The central hypothesis is that the selective translation of mitochondrial proteins observed in liver, skeletal muscle, and heart of mammals during periods of energetic stress is dictated by the complexity of mRNA sequence elements and/or subcellular localization to the mitochondrial reticulum. This hypothesis has been formulated based on evidence generated in the applicants' laboratories as well as extensive published evidence in lower eukaryotes. The rationale for the proposed research is that the successful completion of this research would provide new opportunities to identify therapeutic targets to slow aging and prolong healthspan by targeting mechanisms that have been virtually unstudied in mammalian cells. The research has two specific aims: 1) to determine in vitro the mechanisms responsible for the preferential translation and synthesis of mammalian mitochondrial proteins during the signaling of energetic stress, and the extent of the contribution of autophagy/mitophagy to these increases, and 2) to identify strategies that activate pathways of energetic stress in vivo to preserve mitochondrial protein synthesis and slow aging. To accomplish these specific aims, multiple cell types and tissues will be analyzed. Methods used in vitro include polysome profiles with analysis of rapidly translating mRNA, bioinformatic analysis of mRNA sequences, and novel stable isotope techniques for the determination of mitochondrial protein synthesis and autophagic flux. Methods used in vivo include additional novel isotope techniques for measuring protein synthesis at the tissue, organelle, and individual protein level as well as cellular proliferation. The contribution of the proposed research is significant because it is designed to elucidate an important, but unstudied, regulatory step of mitochondrial biogenesis. Further, it is designed to translate in vitro mechanisms to in vivo studies to determine if these mechanisms can be targeted therapeutically. The proposed research is innovative because it considers post-transcriptional mechanisms of mitochondrial biogenesis, which have previously been largely overlooked. Further, the laboratory is among the very few groups capable of making long-term protein synthetic and cellular proliferation measurements that allow for the translation of in vitro studie to in vivo outcomes. Finally, the in vivo assessment of the synthesis of individual mitochondrial proteins has until now not been feasible, thus the project is technically innovative. The results are expected to have a positive impact because they focus on mechanisms that may offer novel therapeutic targets that are alternatives to a therapy (lifelong caloric restriction) that has provn efficacy, but extremely limited adherence in humans.