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

描述（由申请人提供）：目前没有治愈超过三十种类型的遗传性肌营养不良症，影响世界各地的人们。这些疾病的共同特征是进行性肌无力和肌力丧失。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差异很大。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，但有关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参与肌营养不良和萎缩性肌肉的分子参与者。我们的长期目标是了解负责肌肉蛋白质周转的分子事件，以及这一过程的失调如何导致肌肉营养不良和萎缩。本申请的总体目标是使用遗传上易处理的生物体黑腹果蝇（Drosophila melanogaster）来开发肌肉萎缩疾病，特别是肢带肌营养不良2 H型（LGMD 2 H）的体内模型。我们这个项目的基本原理是LGMD 2 H是由肌肉表达的E3-泛素连接酶蛋白TRIM 32突变引起的。利用果蝇，我们发现了一个类似的基因，dTRIM 32，它在胚胎和成熟的肌肉组织中表达。果蝇中可用的遗传和分子工具使其成为模拟人类疾病的理想生物体，因为果蝇提供比脊椎动物基因组中更短的世代时间和更少的功能冗余。我们有丰富的经验，利用果蝇遗传学来揭示基因功能，我们已经开发了一种体内生物化学/质谱方法，用于识别dTRIM 32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）在成肌细胞融合和成体肌肉结构中表征dTRIM 32缺陷果蝇的表型; 2）确定dTRIM 32的哪些区域对于肌肉功能和/或肌原纤维结构是重要的;和3）通过质谱鉴定和表征发育中的胚胎肌肉和成体肌肉中果蝇E3-泛素连接酶dTRIM 32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营养不良症相关的触发因素和由此产生的生理变化，但有关这些疾病的分子基础的细节才刚刚出现。本文提出的研究旨在更好地了解肌肉蛋白质周转以及该过程的失调如何导致肌肉萎缩。在开发真正成功的治疗策略之前，必须首先更好地了解参与肌营养不良和萎缩性肌肉的分子参与者。

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.