Cullin mediated protein turnover in skeletal and cardiac muscles

7.项目摘要/摘要 库林介导的骨骼肌和心肌蛋白质周转 蛋白质的受控降解是细胞生存的重要机制。积累 琼脂体/包涵体中未降解的蛋白质，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最多四种降解肌纤毛的蛋白分解系统 蛋白质：半胱氨酸蛋白酶和钙蛋白酶、泛素-蛋白酶体系统(UPS)和自噬 系统。大多数细胞蛋白质的降解是通过UPS实现的，需要标记 通过酶的级联反应，通过泛素(多泛素化)底物蛋白质。底物识别 其随后的泛素化涉及E3-泛素连接酶和E2-泛素的协同作用 结合酶。有趣的是，参与肌肉蛋白质泛素化的几种成分是 定位于肌原纤维，类似于Murf蛋白家族的肌肉特异性E3-连接酶。虽然相当可观 人们的注意力一直集中在这些肌肉特异的UPS组件上，关于其作用的信息有限 比较普遍表达的E3-连接酶在肌肉蛋白质周转中的作用，以及它们是否可能发挥更多的作用 在肌病发展和进展中的因果作用。 在研究肌原纤维蛋白的Oblcurin家族时，我们发现了几个先前 与泛素E3连接酶的一个大家族--cullin环蛋白的关联尚未确定。而且我们发现， 其中一些cullin蛋白表现出肌原纤维定位，类似于肌肉特异的E3连接酶。 这些耐人寻味的结果，以及观察到在扩张的模型中剔除环状连接酶发生了变化 心肌病(MLP基因敲除小鼠)和骨骼肌病(暗黑蛋白基因敲除)将我们引向 CULLIN-3及其相关蛋白在肌肉蛋白质周转中的作用可能大于 之前已经预料到了。 这项拟议的研究项目调查了cullin蛋白对肌肉特异性蛋白的生物学作用。 周转率，以及它们与Oblcurin蛋白家族的肌原纤维蛋白的联系，通过体内的组合 分析心肌和骨骼肌生理和功能的方法，使用体外方法 在分子水平上表征cullin-3、其结合伙伴以及它们与黑暗素蛋白的相互作用。 初步研究表明，该项目的结果可能不仅对骨骼肌有影响 肌病，如肢体带状肌营养不良症(2J型)，通过与Oblcurin蛋白的联系，但也用于 心肌病，因为cullin蛋白及其一些结合伙伴在动物中发生了显著的变化 扩张型心肌病模型(MLP基因敲除)。

7. Project Summary/Abstract 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).