Quantitating Mitophagy in Skeletal Muscle Models

描述（由申请人提供）：线粒体自噬是一种特殊形式的巨自噬，负责从细胞中去除无功能的线粒体。鉴于线粒体在氧化应激中发挥的重要作用，有人认为线粒体自噬对骨骼肌中的蛋白质周转至关重要。目前不可能确定每种形式的自噬，包括线粒体自噬，如何促进蛋白质周转。本申请的直接目标是开发生物分析方法，以确定细胞培养物和骨骼肌中的线粒体自噬通量。测量的主要参数是线粒体自噬通量（线粒体自噬发生的速率），使用基于毛细管电泳激光诱导荧光检测（CE-LIF）和蛋白质组学图谱的单个细胞器测量的新技术。长期目标是应用这些生物分析方法来表征 线粒体自噬在人类疾病如甲状腺功能亢进和衰老中的作用，其特征在于蛋白质周转的改变。 本申请的具体目的是：1.定义新的线粒体自噬通量分析的基础上，个别细胞器的测量CE-LIF。2.确定与线粒体自噬通量变化相关的细胞器特异性蛋白质组丰度、羰基化和泛素化的变化。3.测定甲亢大鼠、衰老大鼠和衰老人模型骨骼肌线粒体自噬流量的变化。这项工作从一个独特的角度接近线粒体自噬，旨在整合个体细胞器测量和蛋白质组谱，以定义具有高度特异性的改变的线粒体自噬通量，即使在存在其他形式的自噬的情况下。我们将把这些新技术应用于临床研究，以研究人类骨骼肌中的线粒体自噬。这项工作结果的长期影响将是研究线粒体自噬机制及其在疾病和衰老中的作用的基础。

DESCRIPTION (provided by applicant): Mitophagy is a specialized form of macroautophagy responsible for removal of non-functional mitochondria from the cell. Given the prominent role that mitochondria play in oxidative stress, it has been suggested that mitophagy is critical to protein turnover in skeletal muscle. It is currently impossible to determine how each form of autophagy, including mitophagy, contributes to protein turnover. The immediate goal of this application is to develop bioanalytical approaches to determine mitophagy fluxes in cell cultures and skeletal muscle. The main parameter to measure is mitophagy flux (the rate at which mitophagy occurs) using novel technologies based on individual organelle measurements by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and proteomic profiles. The long-term goal is to apply these bioanalytical approaches to characterize the role of mitophagy in human conditions such as hyperthyroidism and aging that are characterized by alterations in protein turnover. The specific aims of this application are: 1. Define new mitophagy flux analyses based on individual organelle measurements by CE-LIF. 2. Identify organelle-specific proteome changes in abundance, carbonylation and ubiquitination that are associated with changes in mitophagy flux. 3. Determine changes in mitophagy flux in skeletal muscle of hyperthyroid rat, aging rat and aging human models. This work approaches mitophagy from a unique angle, aiming to integrate individual organelle measurements and proteome profiles to define altered mitophagy fluxes with high specificity, even when in the presence of other forms of autophagy. We will the transfer the new technologies to a clinical research setting to investigate mitophagy in human skeletal muscle. Long-term impact of the findings of this work would be the basis for investigating mechanisms of mitophagy and its role in disease and aging.