Unraveling the regulation of MuRF1 activity, a central hub in a dynamic machinery thatpromotes skeletal muscle atrophy.

由于身体紧张、营养不良或其他因素而导致的骨骼肌质量损失对患病或老年人来说是一种健康威胁。因此，降低肌肉萎缩率被认为可以减轻患者的发病率和死亡率。在肌肉萎缩（也称为肌肉萎缩）期间，肌纤维内称为肌节的高度有序的功能结构被降解，该过程涉及泛素蛋白酶体系统（UPS）。事实上，UPS的两个真正的组成部分，“肌肉环指1”（MuRF 1;由Trim 63编码）和Atrogin-1（由Fbxo 32编码）已经被鉴定，这两个都是专门在肌肉细胞中表达，并且这两个都被证明是萎缩过程所需的。尽管如此，对MuRF 1和Atrogin-1的细胞功能及其在肌肉萎缩期间的分子活性知之甚少。在最近的一项工作中，我们确定MuRF 1作为cullin型泛素连接酶的一个组成部分。这些多酶蛋白质复合物围绕cullin支架单元构建，其结合底物募集因子和RING蛋白的不同组合。由于它们的模块化特征，cullin型泛素连接酶介导高度多样化的客户蛋白的泛素化，并通过替换单个底物结合亚基来使它们的活性适应不断变化的要求。各种高通量筛选导致鉴定推定的MuRF 1底物，如肌球蛋白重链（MyHC）、肌球蛋白轻链2、肌联蛋白、肌肉型肌酸激酶和糖皮质激素调节元件结合蛋白-1，但目前尚不清楚这些蛋白的泛素化是否涉及MuRF 1。这项研究计划旨在更好地表征MuRF 1在骨骼肌萎缩中的功能。我们将分析现有的高通量转录组学和蛋白质组学数据，研究萎缩过程中MuRF 1和已知伴侣蛋白的调控，并确定新的相互作用因子以及底物蛋白。然后，我们希望采用生物化学方法来研究这些蛋白质在细胞培养中的功能。然后将在肌肉萎缩的动物系统中研究所选候选物的活性和调节。最后，我们计划用纯化的组分重构含MuRF 1的蛋白质复合物，并在体外监测其泛素化活性。我们的研究结果的生物学意义将使用患者材料进行验证。这项工作将为骨骼肌萎缩期间MuRF 1及其伴侣蛋白的分子活性提供更深入的见解，并可能揭示临床干预的新切入点。

Loss of skeletal muscle mass in response to reduced physical strain, malnutrition, or other factors is a health threat for diseased or elderly people. Therefore, decreasing the rate of muscle wasting is considered to alleviate the morbidity and mortality of patients. During muscle wasting, also termed muscle atrophy, a highly ordered functional structure within myofibers called the sarcomere is degraded and this process involves the ubiquitin proteasome system (UPS). Indeed, two bona fide components of the UPS, the “Muscle RING Finger 1” (MuRF1; encoded by Trim63) and Atrogin-1 (encoded by Fbxo32) have been identified, which both are exclusively expressed in muscle cells and which both were shown to be required for the atrophic process. Still, relatively few is known about the cellular function of MuRF1 and Atrogin-1 and their molecular activities during muscle wasting. In a recent work, we identified MuRF1 as a component of a cullin-type ubiquitin ligase. These multi-enzyme protein complexes are built around a cullin scaffold unit, which binds different combinations of substrate recruitment factors and a RING protein. Due to their modular character, cullin-type ubiquitin ligases mediate the ubiquitination of highly diverse client proteins and adapt their activity to changing requirements by replacing individual substrate binding subunits. Various high-throughput screens led to the identification of putative MuRF1 substrates such as myosin heavy chain (MyHC), myosin light chain 2, titin, muscle-type creatine kinase, and glucocorticoid modulatory element binding protein-1, but it is unclear, whether ubiquitination of these proteins involves MuRF1. This research plan aims to better characterize the function of MuRF1 in skeletal muscle atrophy. We will analyze already available high-throughput transcriptomics and proteomics data to study the regulation of MuRF1 and known partner proteins during atrophy and identify novel interacting factors as well as substrate proteins. We then want to employ biochemical methods to investigate the function of these proteins in cell culture. The activity and regulation of selected candidates will then be studied in an animal system for muscle atrophy. Finally, we plan to reconstitute MuRF1 containing protein complexes with purified components and monitor their ubiquitination activity in vitro. The biological significance of our findings will then be verified using patient material. This work should provide deeper insights into the molecular activities of MuRF1 and its partner proteins during skeletal muscle atrophy and possibly unravel novel entry points for clinical intervention.