The Role of Protein Turnover in a Drosophila Model of Muscle Atrophy.

描述(申请人提供)：目前，全球范围内有30多种遗传性肌营养不良症尚无治愈方法。这些疾病的共同特征是进行性肌肉无力和肌肉力量丧失。然而，疾病的进展在受影响的肌肉、严重程度和发病年龄方面有很大的差异。虽然已经进行了许多研究来检查与肌肉萎缩相关的触发因素和由此产生的生理变化，但关于这些疾病的分子基础的细节才刚刚出现。在开发真正成功的治疗策略之前，必须首先更好地了解肌肉营养不良和肌肉萎缩所涉及的分子因素。我们的长期目标是了解导致肌肉蛋白质周转的分子事件，以及这一过程的错误调节如何导致肌肉营养不良和萎缩。这项应用的总体目标是使用遗传易驯化的黑腹果蝇来建立肌肉消耗性疾病的体内模型，特别是肢体-Girdle肌营养不良2H型(LGMD2H)。我们这个项目的基本原理是LGMD2H是由肌肉表达的E3-泛素连接酶蛋白TRIM32的突变引起的。利用果蝇，我们发现了一个类似的基因dTRIM32，它在胚胎和成熟的肌肉组织中都有表达。苍蝇可用的遗传和分子工具使其成为模拟人类疾病的理想有机体，因为与脊椎动物基因组相比，苍蝇提供的世代时间更短，功能冗余更少。我们有利用果蝇遗传学来揭示基因功能的丰富经验，我们已经开发出一种体内生化/质谱学方法来鉴定dTRIM32的潜在底物。因此，我们计划通过追求以下三个具体目标来实现我们的目标：1)确定dTRIM32缺陷果蝇在成肌细胞融合和成体肌肉结构中的表型特征；2)确定dTRIM32的哪些区域对肌肉功能和/或肌原纤维结构重要；3)通过质谱学鉴定和表征果蝇E3-泛素连接酶dTRIM32在发育中的胚胎肌肉和成体肌肉中的新靶底物。 公共卫生相关性：虽然已经进行了许多研究来检查与肌肉营养不良相关的触发因素和由此产生的生理变化，但关于这些疾病的分子基础的细节才刚刚出现。在此提出的研究旨在更好地了解肌肉蛋白质周转以及这一过程的不当调节如何导致肌肉萎缩。在开发真正成功的治疗策略之前，必须首先更好地了解肌肉营养不良和肌肉萎缩所涉及的分子因素。

DESCRIPTION (provided by applicant): There are currently no cures for over thirty types of inherited muscular dystrophies that affect people worldwide. The common feature in these diseases is progressive muscle weakness and loss of muscle strength. However, progression of the disease varies considerably in the muscles affected, severity, and age of onset. While many studies have been done to examine the triggers and resulting physiological changes associated with muscle wasting, details concerning the molecular basis of these diseases are only emerging. A better understanding of the molecular players involved in muscular dystrophies and atrophic muscle must first be established before truly successful therapeutic strategies can be developed. Our long term goal is to understand the molecular events responsible for muscle protein turnover and how misregulation of this process results in muscular dystrophies and atrophy. The overall objective of this application is to use the genetically tractable organism Drosophila melanogaster to develop an in vivo model for muscle wasting diseases, specifically Limb-Girdle Muscular Dystrophy type2H (LGMD2H). Our rationale for this project is that LGMD2H is caused by a mutation in the muscle-expressed E3-ubiquitin ligase protein TRIM32. Using the fruit fly, we have found a similar gene, dTRIM32, which is expressed in both embryonic and mature muscle tissues. The genetic and molecular tools available in the fly make it an ideal organism to model human disease, as the fly offers shorter generation times and less functional redundancy than found in vertebrate genomes. We have extensive experience using Drosophila genetics to uncover gene function and we have developed an in vivo biochemistry/mass spectrometry approach for identifying potential substrates of dTRIM32. We therefore plan to achieve our objective by pursuit of the following three specific aims: 1) to phenotypically characterize dTRIM32 deficient flies in myoblast fusion and adult muscle structure; 2) to determine which regions of dTRIM32 are important for muscle function and/or myofibril structure; and 3) to identify and characterize new target substrates for the Drosophila E3-ubiquitin ligase dTRIM32 in the developing embryonic muscle and adult muscle by mass spectrometry. PUBLIC HEALTH RELEVANCE: While many studies have been done to examine the triggers and resulting physiological changes associated with muscular dystrophies, details concerning the molecular basis of these diseases are only emerging. The studies proposed herein are designed to gain a better understanding of muscle protein turnover and how misregulation of this process may result in muscle atrophy. A better understanding of the molecular players involved in muscular dystrophies and atrophic muscle must first be established before truly successful therapeutic strategies can be developed.