The E3 ubiquitin ligase MuRF1/TRIM63 was identified 20 years ago and suspected to play important roles during skeletal muscle atrophy. Since then, numerous studies have been conducted to decipher the roles, molecular mechanisms and regulation of this enzyme. This revealed that MuRF1 is an important player in the skeletal muscle atrophy process occurring during catabolic states, making MuRF1 a prime candidate for pharmacological treatments against muscle wasting. Indeed, muscle wasting is an associated event of several diseases (e.g., cancer, sepsis, diabetes, renal failure, etc.) and negatively impacts the prognosis of patients, which has stimulated the search for MuRF1 inhibitory molecules. However, studies on MuRF1 cardiac functions revealed that MuRF1 is also cardioprotective, revealing a yin and yang role of MuRF1, being detrimental in skeletal muscle and beneficial in the heart. This review discusses data obtained on MuRF1, both in skeletal and cardiac muscles, over the past 20 years, regarding the structure, the regulation, the location and the different functions identified, and the first inhibitors reported, and aim to draw the picture of what is known about MuRF1. The review also discusses important MuRF1 characteristics to consider for the design of future drugs to maintain skeletal muscle mass in patients with different pathologies.

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.

The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (K_D = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (K_D = 46.7 nM) over monomeric G-actin (K_D = 450 nM). By contrast with actin that did not alter MuRF1–UBE2L3 affinity, binding of MHC to MuRF1 (K_D = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.

Développement de nouvelles stratégies pour limiter la perte de protéines musculaires médiée par MuRF1/ TRIM63: Plusieurs conditions cataboliques (désuétude, cachexie, ou vieillissement) sont caractérisées par une perte de masse et de force musculaire principalement due à une augmentation de la protéolyse des muscles squelettiques. Lorsqu'elle se prolonge dans le temps, cette faiblesse musculaire contribue à la détérioration de la santé des patients, compromet les traitements et est associée à une mortalité accrue. Dans ce contexte, la protéolyse est principalement orchestrée par le système Ubiquitin Proteasome (UPS). Parmi les différents acteurs de l'UPS, MuRF1/TRIM63 est une ubiquitine-ligase E3 de type RING considérée comme un composant clé de ce processus et donc un candidat de choix pour des traitements pharmacologiques. Cependant, MuRF1/TRIM63 est dépourvue d'activité catalytique et dépend de la présence d'enzymes UBE2 (E2) pour cibler les protéines en vue de leur dégradation dépendante du protéasome. A ce jour, aucun traitement efficace pour prévenir ou limiter la fonte musculaire n'a été découvert. Ce projet de thèse visait donc à combler en partie cette lacune en comprenant les mécanismes de l'atrophie musculaire impliquant MuRF1/TRIM63, afin de développer des stratégies pour prévenir ou limiter la perte de protéines musculaires. En combinant des approches in vitro et in cellulo, nous avons caractérisé les interactions entre MuRF1/TRIM63 et certains de ses substrats majeurs (alpha-actine, chaîne lourde de la myosine [MyHC], etc.) Nous avons montré que l'identité du substrat affecte différentiellement l'interaction entre MuRF1 et plusieurs enzymes E2, UBE2L3, UBE2J1 et UBE2J2. Parmi les différentes enzymes E2, UBE2L3 est apparue comme la plus efficace dans les myotubes C2C12 cataboliques, un effet principal étant observé sur la dégradation des protéines contractiles (alpha-actine et MyHC). En conséquence, le knockdown d'UBE2L3 dans les muscles du Tibialis anterior a induit une hypertrophie chez les souris traitées à la dexaméthasone tandis que la surexpression d'UBE2L3 a aggravé l'atrophie musculaire. En ce qui concerne les UBE2J1 et UBE2J2, à l'heure actuelle, nous ne pouvons pas complètement exclure un rôle sur l'atrophie musculaire. Enfin, en réalisant un criblage à haut débit de composés à petites molécules, nous avons trouvé huit inhibiteurs protéine-protéine potentiels pour perturber les couples MuRF1-UBE2J1 ou MuRF1- UBE2J2. Nos résultats démontrent que les mécanismes impliqués dans la dégradation des protéines contractiles sont plus compliqués que prévu, avec une interaction entre MuRF1, une enzyme E2 et le substrat lui-même. Décrypter les mécanismes biochimiques exacts de la dégradation des protéines sarcomériques est donc obligatoire pour développer de futures stratégies thérapeutiques.

Myotonic dystrophy type 1 (DM1) is characterized by altered RNA processing, muscle wasting and metabolic dysregulation. In a previous in vitro study, we found reduced endogenous oleic acid (OA; 18:1n-9) in myogenic cells from DM1 patients, and short-term OA supplementation rescued key disease readouts. Here, we asked whether lipid pathways are perturbed in a DM1 in vivo background. We employed the widely used HSA^LR mouse model, which expresses human skeletal alpha-actin transcripts harbouring expanded CTG repeats and recapitulates key DM1 phenotypes. RNA-seq-derived functional enrichment across three independent HSA^LR datasets revealed a consistent enrichment of downregulated GO terms related to fatty-acid metabolism, suggesting impaired lipid handling. Guided by transcriptomic evidence of fatty-acid pathway downregulation, we quantified endogenous OA in HSA^LR mice versus FVB controls by LC-MS across sex, age (postnatal day 21 and 4 months) and tissue (quadriceps, gastrocnemius, and plasma), reporting values per tissue weight and per total protein. OA showed selective, context-dependent reductions in skeletal muscle, varying with tissue, sex, age and normalization, with the clearest deficits in males and in the gastrocnemius, while no generalized deficit was observed. Plasma OA was transiently higher at P21 in both sexes, consistent with the oleate-rich weaning mice diet. Together, the RNA-seq enrichment signal for fatty-acid pathways and the context-dependent OA changes point to network-level control (substrate supply, partitioning/transport, cofactors or downstream utilization), positioning OA as a potential measurable candidate marker of DM1-like muscle pathology in HSA^LR mice.