MOLECULAR BASIS FOR MUSCLE PROTEIN LOSS IN CACHEXIA

恶病质肌肉蛋白损失的分子基础

• 批准号: 6176116
• 负责人: STEWART H LECKER
• 金额: $ 12.12万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6176116
• 关键词: cachexia; cell free system; chronic renal failure; diabetes mellitus; enzyme substrate; genetically modified animals; laboratory mouse; laboratory rat; molecular pathology; muscle metabolism; muscle proteins; myofibrils; proteasome; proteolysis; ubiquitin

Muscle wasting, which occurs mainly by an activation of the ubiquitin- proteasome degradative pathway, is a prominent, debilitating feature of many disease states, including diabetes mellitus and renal failure. Recently, using a newly established cell-free system, we have been able to demonstrate that rates of ubiquitin (Ub) conjugation increase in atrophying muscles from septic; tumor-bearing, diabetic and uremic rats, and that a subset of Ub conjugating enzymes, the N-end rule pathway, is responsible for most of the enhanced Ub conjunction in these atrophying muscles. This is an interesting, unexpected discovery because the N-end rule pathway has been viewed as a minor ubiquitination system that was only involved in the elimination of certain abnormal polypeptides. These results raise the possibility that in cachexia, muscle proteins may be modified to become substrates for this pathway. We propose to use our newly developed cell-free system to further characterize this process. We will measure the abundance and activity of the N-end rule pathway enzymes (E1, E2/14K, and E3alpha) to identify the ones which are responsible for the enhanced proteolysis, and identify the substrates in muscle for these enzymes. In collaborative studies, we will genetically produce animals in which these enzymes are deleted to directly show their requirement in muscle atrophy. Finally, since most of the loss of muscle protein during muscle atrophy is from myofibrillar components, we will begin to study how the myofibril may serve as a source of substrates of the Ub-proteasome pathway by developing an assay for myofibril disassembly. Defining the components of the Ub-proteasome pathway and myofibril disassembly which are modulated in diabetes and renal failure should not only help to illuminate the regulation of muscle protein turnover, but also may allow the development of inhibitors that could combat the morbidity of these catabolic diseases. These studies will be performed in the laboratory of Dr. Alfred Goldberg, a leader in the fields of muscle proteolysis and the Ub- proteasome pathway. The applicant is a graduate of the M.D./Ph.D. program at UCLA, completing a Nephrology fellowship at the Beth Israel Deaconess Medical Center and Harvard Medical School. His long-term goal is to develop a research program centered on problems of protein folding and degradation relevant to kidney disease. This proposal offers the unique opportunity for the applicant to obtain further cell biology training, gaining experience in animal physiology, DNA technology, and biochemistry, while studying clinically relevant problems in renal disease.

肌肉萎缩主要是由于泛素-蛋白酶体降解途径的激活而发生的，是许多疾病状态（包括糖尿病和肾衰竭）的一个突出的、使人衰弱的特征。最近，使用新建立的无细胞系统，我们已经能够证明泛素 (Ub) 结合率在脓毒症引起的萎缩肌肉中增加；患有肿瘤、糖尿病和尿毒症的大鼠，并且 Ub 结合酶的一个子集（N 端规则途径）负责这些萎缩肌肉中 Ub 结合的大部分增强。这是一个有趣的、意想不到的发现，因为N端规则途径一直被视为一个次要的泛素化系统，仅涉及某些异常多肽的消除。这些结果提出了这样的可能性：在恶病质中，肌肉蛋白可能被修饰成为该途径的底物。我们建议使用我们新开发的无细胞系统来进一步表征这一过程。我们将测量 N 端规则途径酶（E1、E2/14K 和 E3alpha）的丰度和活性，以确定负责增强蛋白水解的酶，并确定肌肉中这些酶的底物。在合作研究中，我们将通过基因技术培育出这些酶被删除的动物，以直接显示它们对肌肉萎缩的需求。最后，由于肌肉萎缩期间肌肉蛋白的大部分损失来自肌原纤维成分，因此我们将通过开发肌原纤维分解测定法来开始研究肌原纤维如何作为 Ub 蛋白酶体途径的底物来源。定义在糖尿病和肾功能衰竭中受到调节的 Ub 蛋白酶体途径和肌原纤维解体的组成部分不仅有助于阐明肌肉蛋白质周转的调节，而且还可能允许开发能够对抗这些分解代谢疾病的发病率的抑制剂。这些研究将在 Alfred Goldberg 博士的实验室中进行，他是肌肉蛋白水解和 Ub-蛋白酶体途径领域的领导者。申请人是医学博士/博士毕业生。加州大学洛杉矶分校的项目，在贝斯以色列女执事医疗中心和哈佛医学院完成肾脏病学研究金。他的长期目标是开发一个以与肾脏疾病相关的蛋白质折叠和降解问题为中心的研究项目。该提案为申请人提供了获得进一步细胞生物学培训的独特机会，获得动物生理学、DNA 技术和生物化学方面的经验，同时研究肾脏疾病的临床相关问题。