MOLECULAR BASIS FOR MUSCLE PROTEIN LOSS IN CACHEXIA

肌肉萎缩主要通过激活泛素-蛋白酶体降解途径发生，是许多疾病状态（包括糖尿病和肾功能衰竭）的一个突出的衰弱特征。最近，使用新建立的无细胞系统，我们已经能够证明，泛素（Ub）结合率在脓毒性萎缩肌肉中增加；在这些萎缩的肌肉中，Ub结合酶的一个亚群，即n端规则通路，负责大部分增强的Ub结合。这是一个有趣的，意想不到的发现，因为n端规则通路被视为一个次要的泛素化系统，只参与某些异常多肽的消除。这些结果提出了在恶病质中，肌肉蛋白可能被修饰成为该途径的底物的可能性。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质水解的酶，并确定这些酶在肌肉中的底物。在合作研究中，我们将从基因上培育出这些酶被删除的动物，以直接显示它们在肌肉萎缩中的需求。最后，由于肌肉萎缩过程中肌肉蛋白的大部分损失来自肌原纤维成分，我们将开始研究肌原纤维如何作为ub蛋白酶体途径的底物来源，通过开发肌原纤维分解的测定。确定ub -蛋白酶体途径和肌原纤维分解在糖尿病和肾衰竭中被调节的成分不仅有助于阐明肌肉蛋白转换的调节，而且可能允许开发抑制剂来对抗这些分解代谢疾病的发病率。这些研究将在Alfred Goldberg博士的实验室进行，他是肌肉蛋白水解和Ub-蛋白酶体途径领域的领导者。申请人是医学博士毕业生。并在贝斯以色列女执事医疗中心和哈佛医学院完成了肾脏病学研究。他的长期目标是发展一个研究项目，重点研究与肾脏疾病相关的蛋白质折叠和降解问题。该申请为申请人提供了一个独特的机会，可以获得进一步的细胞生物学培训，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。

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.