Exploring the function of 1110002E22Rik/C4orf54, a novel target of Fbxo30/MUSA1 during muscle wasting

Skeletal muscle is the most abundant tissue in the human body. Moreover, skeletal muscle health and function are guaranteed by a finely tuned balance between protein synthesis and protein degradation. The disruption of this equilibrium, with protein degradation that overcomes protein synthesis, leads to a pathological condition known as muscle atrophy. One of the major proteolytic systems present in mammals is the ubiquitin-proteasome system (UPS), in which E3 ubiquitin ligases coordinate specific conjugation of ubiquitin moieties onto target proteins en route for degradation. Importantly, in the past few years, Sandri's laboratory has discovered a previously uncharacterized E3 ubiquitin ligase, Fbxo30/MUSA1. Notably, MUSA1 stands at the crossroads of the two major pathways regulating muscle mass; indeed, its expression is suppressed by the BMP-Smad1/5/8 axis, while FoxO3 transcription factor directly controls its expression under catabolic conditions. Understanding MUSA1 function in skeletal muscle physiology is therefore crucial. To do this, a constitutive muscle-specific MUSA1 knockout mouse model was generated. MUSA1 deficient muscles did not show any pervasive phenotype in adult mice. However, electron microscopy data showed massive accumulation of undigested proteins in MUSA1 knockout muscles that underwent two weeks of surgical denervation, and at 22-23 months of age, appearing as electron-dense infiltrates completely disrupting sarcomeres organization. To better decipher cellular events leading to the observed phenotype, proteomics analyses were performed. Interestingly, both denervated and aged MUSA1 knockout muscles showed increased levels of several cytoskeleton-related proteins, actin-binding proteins and Z-disc components. These results suggest that MUSA1 could be responsible for some of these structural proteins' ubiquitination-mediated degradation, directly or indirectly. Among the most significantly upregulated proteins in MUSA1 knockout muscles proteomes, the presence of a previously uncharacterized protein, 1110002E22Rik (human homolog: C4orf54), encoded by a gene belonging to the Riken cDNAs collection (RIKENs), was noticed. Given the importance of exploring the so-called "neglectome", and since bioinformatic predictions show that 1110002E22Rik gene encodes a ≈ 190 kDa protein exclusively expressed in skeletal muscle and myocardium, Sandri's laboratory was encouraged to start its further characterization. Indeed, this thesis focuses on a preliminary molecular characterization of 1110002E22Rik, aiming to provide preliminary insights into its role in skeletal muscle physiology, as well as to investigate its relationship with Fbxo30/MUSA1.