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

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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.