Quantitating Mitophagy in Skeletal Muscle Models

描述（由申请人提供）：线粒体自噬是巨噬的一种特殊形式，负责从细胞中去除非功能性线粒体。鉴于线粒体在氧化应激中所起的突出作用，有人认为线粒体自噬对骨骼肌中的蛋白质周转至关重要。目前还不可能确定每种形式的自噬（包括有丝自噬）如何促进蛋白质周转。本应用程序的直接目标是开发生物分析方法，以确定细胞培养和骨骼肌中的有丝分裂通量。测量的主要参数是线粒体自噬通量（发生线粒体自噬的速率），使用基于单个细胞器测量的新技术，通过毛细管电泳和激光诱导荧光检测（CE-LIF）和蛋白质组学谱。长期目标是应用这些生物分析方法来描述

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.