Atrogin-1 and Muscle Protein Balance

描述(申请人提供)：肌肉萎缩和负氮平衡是许多疾病的特征，包括糖尿病，慢性肾功能衰竭和癌症。我们最近发现了一个新的基因，阿托金-1，它在骨骼肌中特异表达，并且在这些疾病的各种动物模型中肌肉萎缩时被强烈诱导。阿托金-1蛋白含有一个F-box，这表明它可能作为一种泛素蛋白连接酶，催化关键蛋白的泛素化和降解，导致肌肉萎缩。这项建议是在阿尔弗雷德·戈德堡博士的实验室发起的研究的结果，莱克博士在那里是博士后研究员，现在将主要在莱克博士的独立实验室继续进行。这种新蛋白质的功能和重要性将通过结合遗传和生化方法来阐明。培养中的肌肉细胞将被工程诱导表达萎缩素-1，以便研究其对肌肉生长、分化和萎缩的影响。小鼠的萎缩素-1基因将被破坏，以产生一种基因敲除菌株，在那里我们可以测试缺乏萎缩素-1的小鼠是否会出现肌肉萎缩。为了了解阿托金-1是如何促进肌肉蛋白质分解的，我们将尝试通过酵母双杂交方法和阿托金-1相关蛋白的生化分离来确定其靶点和辅因子。最后，我们将开发体外实验来测量阿托金-1结合泛素到蛋白质的能力。阐明阿托金-1的功能不仅有助于表征肌肉蛋白质分解的机制和生理调节，而且可能有助于开发药物抑制剂来对抗肌肉萎缩及其相关的发病率(例如，肾功能衰竭中的尿毒症)。

DESCRIPTION (provided by applicant): Muscle wasting and negative nitrogen balance are debilitating features of many disease states including diabetes, chronic renal failure and cancer. We have recently identified a new gene, atrogin-1, which is expressed specifically in skeletal muscle and is strongly induced when muscle atrophies in various animal models of these diseases. The atrogin-1 protein contains an F-box, suggesting that it may act as a ubiquitin-protein ligase that catalyzes the ubiquitination and degradation of key proteins, leading to muscle wasting. This proposal is an outgrowth of studies which were initiated in Dr. Alfred Goldberg's laboratory, where Dr. Lecker was a postdoctoral fellow, and will now be continued mainly in the independent laboratory of Dr. Lecker. The functions and importance of this novel protein will be clarified by utilizing a combination of genetic and biochemical approaches. Muscle cells in culture will be engineered to inducibly express atrogin-1, so that its effects on muscle growth, differentiation and atrophy can be studied. The mouse atrogin-1 gene will be disrupted to generate a knockout strain where we can test whether mice lacking atrogin-1 can develop muscle atrophy. In an effort to understand how atrogin-1 might promote muscle protein breakdown, we will attempt to identify its targets and cofactors by the yeast two-hybrid approach and biochemical isolation of atrogin-1-associated proteins. Finally, we will develop in vitro assays to measure the ability of atrogin-1 to conjugate ubiquitin to proteins. Elucidating the functions of atrogin-1 will not only help characterize the mechanisms and physiological regulation of muscle protein breakdown but may also allow the development of pharmacological inhibitors that could combat muscle wasting conditions and its associated morbidity (e.g. the uremia in renal failure).