Cullin mediated protein turnover in skeletal and cardiac muscles

描述（由申请人提供）：Cullin介导的骨骼肌和心肌中的蛋白质周转，蛋白质的调节降解是细胞存活的重要机制。聚合体/包涵体中未降解蛋白质的积累或（突变）蛋白质的过早降解通常与疾病的发展有关，如心脏病和骨骼肌肌病。心脏和骨骼肌细胞含有多达四种降解肌纤维蛋白的蛋白水解系统：半胱天甲酶和钙蛋白酶，泛素蛋白酶体系统（UPS）和自噬系统。大多数细胞蛋白的降解是通过UPS的方式实现的，并且需要通过酶级联用泛素（多泛素化）标记底物蛋白。底物的识别及其随后的泛素化涉及e3 -泛素连接酶和e2 -泛素偶联酶的协同作用。有趣的是，参与肌肉蛋白泛素化的几个组分沿着肌原纤维定位，如MuRF蛋白家族的肌肉特异性e3连接酶。虽然大量的注意力集中在这些肌肉特异性的UPS成分上，但关于更普遍表达的e3连接酶在肌肉蛋白周转中的作用，以及它们是否可能在肌病的发生和进展中发挥更大的因果作用的信息有限。在研究肌纤维蛋白的obscurin家族时，我们发现了几个以前未被表征的与cullin-RING蛋白的连接，cullin-RING蛋白是一个泛素e3连接酶的大家族。此外，我们发现其中一些cullin蛋白表现出肌纤维定位，与肌肉特异性e3连接酶相当。这些有趣的结果，以及cullin-RING连接酶在扩张型心肌病（MLP敲除小鼠）和骨骼肌肌病（obscurin敲除小鼠）模型中发生改变的观察，使我们提出cullin-3及其相关蛋白可能在肌肉蛋白转换中发挥比先前预期更大的作用的假设。拟议的研究项目通过结合体内方法分析心脏和骨骼肌生理学和功能，以及体外方法表征cullin-3及其结合伙伴及其在分子水平上与obscurin蛋白的关联，研究cullin蛋白在肌肉特异性蛋白质周转中的生物学作用，以及它们与obscurin蛋白家族的肌纤维蛋白的联系。初步研究表明，该项目的结果可能不仅对骨骼肌肌病（如肢带肌营养不良症（2J型））有影响，而且对心肌病也有影响，因为cullin蛋白及其一些结合伙伴在扩张型心肌病（MLP敲除）的动物模型中显着改变。

DESCRIPTION (provided by applicant): Cullin mediated protein turnover in skeletal and cardiac muscles The regulated degradation of proteins is an important mechanism for the survival of cells. Accumulation of undegraded proteins in aggrosomes/inclusion bodies, or the premature degradation of (mutant) proteins is often associated with development of diseases, like cardiac and skeletal muscle myopathies. Cardiac and skeletal muscle cells contain up to four proteolytic systems for the degradation of myofbrillar proteins: the caspase and calpain proteases, the ubiquitin-proteasome system (UPS), and the autophagy system. Degradation of most cellular proteins is achieved by way of the UPS and requires tagging of substrate proteins by ubiquitin (poly-ubiquitylation) through an enzymatic cascade. Recognition of substrate and its subsequent ubiquitylation involves the concerted action of E3-ubiquitin ligases and E2-ubiquitin conjugating enzymes. Intriguingly, several components involved in the ubiquitylation of muscle proteins are localized along myofibrils, like the muscle specific E3-ligases of the MuRF protein family. While considerable attention has been focused on these muscle specific UPS components, there is limited information on the role of the more ubiquitously expressed E3-ligases in muscle protein turnover, and whether they may play a more causal role for the development and progression of myopathies. While investigating the obscurin family of myofibrillar proteins, we identified several previously uncharacterized links to cullin-RING proteins, a large family of ubiquitin E3-ligases. Moreover we found that some of these cullin proteins exhibit a myofibrillar localization, comparable to muscle specific E3-ligases. These intriguing results, and the observation that cullin-RING ligases are altered in models for dilated cardiomyopathy (MLP knockout mouse) and skeletal muscle myopathy (obscurin knockout) led us to the hypothesis that cullin-3 and its associated proteins may play a greater role for muscle protein turnover than previously anticipated. The proposed research project investigates the biological role of cullin proteins for muscle specific protein- turnover, and their links to myofibrillar proteins of the obscurin protein family, through a combination of in vivo approaches that analyze cardiac and skeletal muscle physiology and function, with in vitro methods that characterize cullin-3, its binding partners, and their association to obscurin proteins at the molecular level. Preliminary studies indicate that results from this project may have ramifications not only for skeletal muscle myopathies, like the limb-girdle muscular dystrophy (type 2J) through links with obscurin proteins, but also for cardiomyopathies, since cullin proteins, and some of their binding partners are significantly altered in animal models for dilated cardiomyopathy (MLP knockout). PUBLIC HEALTH RELEVANCE: The project "Cullin mediated protein turnover in skeletal and cardiac muscles" investigates the biological function of cullin proteins, a class of enzymes that is important for protein degradation. Particularly the role of cullin-3, and its binding partners, for the proper development and physiological function for heart and skeletal muscles will be analyzed. Preliminary studies indicate that results from this project may have ramifications not only for skeletal muscle myopathies, like the limb-girdle muscular dystrophy (type 2J) through links with obscurin proteins, but also for cardiomyopathies, since cullin proteins, and some of their binding partners are significantly altered in animal models for dilated cardiomyopathy (MLP knockout).