Cullin mediated protein turnover in skeletal and cardiac muscles

描述(由申请人提供)：库林介导骨骼肌和心肌中的蛋白质周转蛋白质的受控降解是细胞生存的重要机制。未降解的蛋白质在集合体/包涵体中积累，或(突变的)蛋白质的过早降解通常与疾病的发展有关，如心肌和骨骼肌病。心肌和骨骼肌细胞含有多达四种降解肌纤蛋白的蛋白分解系统：caspase和calain蛋白水解酶、泛素-蛋白酶体系统(UPS)和自噬系统。大多数细胞蛋白质的降解是通过UPS的方式实现的，需要通过酶的级联作用通过泛素(多泛素化)标记底物蛋白质。底物的识别及其随后的泛素化涉及E3-泛素连接酶和E2-泛素结合酶的协同作用。有趣的是，参与肌肉蛋白泛素化的几个成分沿着肌原纤维定位，例如Murf蛋白家族的肌肉特异性E3-连接酶。虽然这些肌肉特异的UPS成分已经引起了相当多的关注，但关于更普遍表达的E3-连接酶在肌肉蛋白质周转中的作用，以及它们是否在肌病的发生和发展中起到更具因果作用的信息有限。在研究肌原纤维蛋白的Oblcurin家族时，我们发现了几个以前未被描述的与泛素E3连接酶家族--cullin环蛋白的联系。此外，我们还发现其中一些cullin蛋白具有肌原纤维定位，类似于肌肉特异性的E3-连接酶。这些耐人寻味的结果，以及在扩张型心肌病(MLP基因敲除小鼠)和骨骼肌肌病(Darcurin基因敲除)模型中观察到cullin-环连接酶发生变化的情况，使我们提出了这样的假设，即cullin-3及其相关蛋白可能对肌肉蛋白质周转起到比先前预期更大的作用。这项拟议的研究项目通过结合体内分析心肌和骨骼肌生理和功能的方法，以及在体外表征cullin-3、其结合伙伴以及它们与obcurin蛋白之间的关系的方法，研究cullin蛋白在肌肉特定蛋白周转中的生物学作用，以及它们与Oblcurin蛋白家族的肌原纤维蛋白的联系。初步研究表明，该项目的结果可能不仅对骨骼肌病有影响，如通过与obcurin蛋白联系的肢体带状肌营养不良症(2J型)，还可能对心肌病产生影响，因为在扩张型心肌病(MLP基因敲除)的动物模型中，cullin蛋白及其一些结合伙伴发生了显著变化。 与公共健康相关：“cullin介导骨骼肌和心肌中的蛋白质周转”项目研究cullin蛋白的生物学功能，cullin蛋白是一类对蛋白质降解很重要的酶。特别是，对于心脏和骨骼肌的正常发育和生理功能，cullin-3及其结合伙伴的作用将被分析。初步研究表明，该项目的结果可能不仅对骨骼肌病有影响，如通过与obcurin蛋白联系的肢体带状肌营养不良症(2J型)，还可能对心肌病产生影响，因为在扩张型心肌病(MLP基因敲除)的动物模型中，cullin蛋白及其一些结合伙伴发生了显著变化。

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).