Quantitating Mitophagy in Skeletal Muscle Models

描述(申请人提供)：有丝分裂吞噬是一种特殊形式的巨型自噬，负责将无功能的线粒体从细胞中移除。鉴于线粒体在氧化应激中的重要作用，已有研究表明，线粒体吞噬作用对骨骼肌中蛋白质的周转至关重要。目前还不可能确定每种形式的自噬，包括有丝分裂吞噬，如何促进蛋白质周转。这项应用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吞丝分裂通量。要测量的主要参数是基于毛细管电泳法和激光诱导荧光检测(CE-LIF)的单个细胞器测量的新技术和蛋白质组图谱，以测量有丝分裂通量(发生有丝分裂的速率)。长期的目标是应用这些生物分析方法来表征 在人类条件下，如甲亢和衰老，以蛋白质周转变化为特征的有丝分裂。本申请的具体目的是：1.根据CE-LIF对单个细胞器的测量，定义新的有丝分裂通量分析。2.确定细胞器特有的蛋白质组在丰度、羰化和泛素化方面的变化，这些变化与有丝分裂通量的变化有关。3.测定甲亢大鼠、衰老大鼠和衰老人体模型大鼠骨骼肌线粒体吞噬通量的变化。这项工作从一个独特的角度探讨有丝分裂吞噬，旨在整合单个细胞器测量和蛋白质组图谱，以确定改变的有丝分裂吞噬通量具有高特异性，即使在存在其他形式的自噬。我们将把新技术转移到临床研究环境中，以研究人类骨骼肌的有丝分裂现象。这项工作发现的长期影响将是研究有丝分裂的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.