Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics

Abstract

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

Redox Signaling and Sarcopenia: Searching for the Primary Suspect

“…Two of the most important E3 ubiquitin ligases with respect to UPS are Muscle RING-finger protein-1 (MuRF-1) and atrophy gene-1/muscle atrophy f-box (Atrogin-1/MAFbx). These proteins are responsible for the degradation of several myofibrillar, sarcomeric, and related regulatory proteins [8,9]. The UPS is commonly upregulated in many other wasting conditions and is heavily influenced by the forkhead box class O (FOXO) transcription factor family [10].…”

The AAA-ATPase ATAD1 and its partners promote degradation of desmin intermediate filaments in muscle

“…HUWE1, see Table II). Desmin was recently reported to be ubiquitinated by the ubiquitin ligase MURF1 in normal mouse muscles overexpressing Murf1 , though this ubiquitination of desmin did not lead to degradation (Baehr et al, 2021). We could not precipitate MURF1 with ATAD1 from denervated muscle extracts, and it remains to be determined if MURF1 acts on desmin in vivo during atrophy.…”

Recent advances in measuring and understanding the regulation of exercise-mediated protein degradation in skeletal muscle

“…More recently, Baehr et al (66) demonstrated that overexpression of MuRF1, but not MAFbx, is sufficient to induce muscle atrophy in mice. In an attempt to understand how MuRF1 regulates myofibrillar protein degradation, Baehr et al (66) identified MuRF1 overexpression-dependent ubiquitylation sites on 56 proteins.…”

“…More recently, Baehr et al (66) demonstrated that overexpression of MuRF1, but not MAFbx, is sufficient to induce muscle atrophy in mice. In an attempt to understand how MuRF1 regulates myofibrillar protein degradation, Baehr et al (66) identified MuRF1 overexpression-dependent ubiquitylation sites on 56 proteins. Surprisingly, their validation showed that the majority of these MuRF1 substrates do not undergo degradation, which is in contrast to Clarke et al's findings (67) that MuRF1 physically associates with and degrades slow and fast myosin heavy chains under Figure 2.…”

“…This notion was also supported by a recent study led by Goodman et al ( 68) that overexpression of ASB2b not only induced atrophy but also increased expression of ubiquitin E1 and E2 enzymes and other E3 ligases (e.g., MUSA1, the muscle-specific Fbxo40, and MuRF2). Interestingly, Baehr et al (66) also showed that muscle atrophy was prevented when the RING domain of MuRF1 is mutated at C44S/C47S. The RING domain is required for binding with E2 conjugating enzymes and catalyzing the transfer of ubiquitin from E2 to substrates (69).…”