MOLECULAR BASIS FOR MUSCLE PROTEIN LOSS IN CACHEXIA

肌肉萎缩主要是通过激活泛素-蛋白酶体降解途径而发生的，是包括糖尿病和肾功能衰竭在内的许多疾病的一个突出的、使人虚弱的特征。最近，利用一个新建立的无细胞系统，我们已经能够证明，在脓毒症、肿瘤、糖尿病和尿毒症大鼠的萎缩肌肉中，泛素(Ub)结合率增加，并且Ub结合酶的一个子集，即N-端规则途径，负责这些萎缩肌肉中大部分Ub结合的增强。这是一个有趣的、意想不到的发现，因为N-端规则通路一直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为这一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-末端规则通路酶(E1、E2/14K和E3α)的丰度和活性，以确定哪些酶对增强的蛋白分解负责，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质的大部分损失来自肌原纤维成分，我们将通过开发一种肌原纤维分解的方法来研究肌原纤维如何作为Ub-蛋白酶体途径底物的来源。明确在糖尿病和肾功能衰竭中调节的Ub-蛋白酶体途径和肌原纤维分解的成分不仅有助于阐明肌肉蛋白质周转的调节，而且可能有助于开发能够对抗这些分解代谢疾病的发病率的抑制剂。这些研究将在阿尔弗雷德·戈德堡博士的实验室进行，他是肌肉蛋白分解和Ub-蛋白酶体途径领域的领导者。申请者毕业于加州大学洛杉矶分校的医学博士项目，在贝丝以色列女执事医学中心和哈佛医学院完成了一项肾脏学奖学金。他的长期目标是开发一个研究项目，重点研究与肾脏疾病相关的蛋白质折叠和降解问题。这项建议为申请者提供了一个独特的机会，在研究肾脏疾病的临床相关问题的同时，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。

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.