The RING-type E3 ubiquitin-protein ligase MuRF1 (also known as TRIM63) plays an important role in skeletal muscle atrophy by targeting contractile proteins. In cellulo, MuRF1 can alternatively interact with four E2 enzymes (UBE2E1, UBE2J1, UBE2J2, or UBE2L3), suggesting different functions or targets for the four MuRF1-E2 complexes. In this article, we studied the interface of these MuRF1-UBE2 complexes based on AlphaFold2 and AlphaFold3 predictions. These predictions revealed the involvement of different residues at the interface of each complex. We confirmed this overall interface difference by the differential sensitivity of MuRF1-E2 complexes to regenerating solutions in surface plasmon resonance experiments. We further confirmed several predictions individually by affinity measurements with point-mutant E2 enzymes and truncated MuRF1. We used the interaction-induced fluorescence change approach with fluorescent MuRF1. Besides canonical E2-RING-type E3 interactions, we were able to identify selective contact points between MuRF1 and its UBE2 partners. Furthermore, in the case of the MuRF1-E2E1 pair, unlike the other MuRF1-E2 pairs, the interaction may also be governed by a domain outside the RING domain. Since the function of RING-type E3s is regulated by E2 enzymes, deciphering the mechanisms of selective recruitment of E2s by MuRF1 paves the way for the development of targeted therapeutics to fight muscle atrophy.

In order to preserve muscle mass during catabolic states, investigators are actively searching for a specific inhibitor of MuRF1, the only known E3 ligase that can target muscle contractile proteins for their degradation. However, what would be the consequences of such inhibitors on other organs, both in the short and long term? Indeed, skeletal muscles can provide amino acids for liver gluconeogenesis, which is a crucial adaptation for maintaining glucose homeostasis upon elevated energy demands (e.g., during prolonged starvation). Comparing 3-month-old wild-type and MuRF1-KO mice, we measured tissue weights, liver glycogen, lipid and protein content, and liver biochemical composition using Fourier transform infrared (FTIR) spectrometry in control animals and in dexamethasone (Dex)-treated animals. Dex induces a catabolic situation with muscle atrophy and lipid deposits in the liver. In response to Dex treatment, liver glycogen, lipid, and protein content increased in wild type (WT) and MuRF1-KO mice. We found that MuRF1 deletion differentially affected organ weights, the liver of KO mice being hypertrophied upon Dex treatment when compared to WT mice. Upon Dex treatment, muscle mass was preserved in MuRF1-KO mice, and by contrast, liver lipid content increased more in these animals than in WT mice. PLS-DA analysis of FTIR showed that the levels of 13 markers were significantly altered in KO vs WT mice, witnessing profound alterations of lipid, protein, and glycogen content in the liver due to the absence of MuRF1. Using Nile red and oil red lipid staining, we also found that both membrane-linked lipids and intracellular lipid droplets were altered due to the absence of MuRF1. Altogether, it seems that when the liver is deprived of the possibility of obtaining amino acids from muscle upon Dex treatment, there is a concomitant increase in tissue weight and anabolic activity.

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 (KD = 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 (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1–UBE2L3 affinity, binding of MHC to MuRF1 (KD = 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.

AbstractBackgroundLoss of muscle mass worsens many diseases such as cancer and renal failure, contributes to the frailty syndrome, and is associated with an increased risk of death. Studies conducted on animal models have revealed the preponderant role of muscle proteolysis and in particular the activation of the ubiquitin proteasome system (UPS). Studies conducted in humans remain scarce, especially within renal deficiency. Whether a shared atrophying programme exists independently of the nature of the disease remains to be established. The aim of this work was to identify common modifications at the transcriptomic level or the proteomic level in atrophying skeletal muscles from cancer and renal failure patients.MethodsMuscle biopsies were performed during scheduled interventions in early‐stage (no treatment and no detectable muscle loss) lung cancer (LC), chronic haemodialysis (HD), or healthy (CT) patients (n = 7 per group; 86% male; 69.6 ± 11.4, 67.9 ± 8.6, and 70.2 ± 7.9 years P > 0.9 for the CT, LC, and HD groups, respectively). Gene expression of members of the UPS, autophagy, and apoptotic systems was measured by quantitative real‐time PCR. A global analysis of the soluble muscle proteome was conducted by shotgun proteomics for investigating the processes altered.ResultsWe found an increased expression of several UPS and autophagy‐related enzymes in both LC and HD patients. The E3 ligases MuRF1 (+56 to 78%, P < 0.01), MAFbx (+68 to 84%, P = 0.02), Hdm2 (+37 to 59%, P = 0.02), and MUSA1/Fbxo30 (+47 to 106%, P = 0.01) and the autophagy‐related genes CTPL (+33 to 47%, P = 0.03) and SQSTM1 (+47 to 137%, P < 0.01) were overexpressed. Mass spectrometry identified >1700 proteins, and principal component analysis revealed three differential proteomes that matched to the three groups of patients. Orthogonal partial least square discriminant analysis created a model, which distinguished the muscles of diseased patients (LC or HD) from those of CT subjects. Proteins that most contributed to the model were selected. Functional analysis revealed up to 238 proteins belonging to nine metabolic processes (inflammatory response, proteolysis, cytoskeleton organization, glucose metabolism, muscle contraction, oxidant detoxification, energy metabolism, fatty acid metabolism, and extracellular matrix) involved in and/or altered by the atrophying programme in both LC and HD patients. This was confirmed by a co‐expression network analysis.ConclusionsWe were able to identify highly similar modifications of several metabolic pathways in patients exhibiting diseases with different aetiologies (early‐stage LC vs. long‐term renal failure). This strongly suggests that a common atrophying programme exists independently of the disease in human.

Change in author names (order). [...]

Introduction: Muscle atrophy is frequently encountered in diseased patients. It contributes to patient’s frailty and is associated with an increased risk of death. Studies using animal models suggest the involvement of the Ubiquitin Proteasome System (UPS) in renal failure-induced muscle atrophy. However, this remains to be established in humans. Another important goal is to detect markers that may help fighting against muscle atrophy through nutritional or pharmacological strategies. Indeed, it is very difficult to counteract the increased proteolysis when it is established. Our objectives were (i) to identify the proteolytic systems activated in chronic hemodialysis (HD) or lung cancer (LC) patients, i.e. pathologies having a different etiology and (ii) to identify markers specific to the activation of muscle atrophy processes independently of the pathology per se. Methods: Muscle biopsies (n = 7 per group) were obtained upon programmed surgery. mRNA and protein levels were determined using qRT-PCR, immunoblotting and proteomic approaches. Results: We found that the UPS and autophagy were activated in both HD and LC patients. Mass spectrometry analysis identified > 1700 proteins. Main component analysis revealed 3 distinct protein expression profiles corresponding to the 3 groups studied. We identified 106 proteins that were significantly modified (decreased or increased) in both HD and LC patients compared to controls (CT). Hierarchical cluster analysis showed that expression levels of these proteins distinguished diseased (HD or LC) vs. CT patients. Orthogonal partial least square discriminant analysis confirmed these results. Conclusion: We demonstrated that the UPS and autophagy were activated during long-term disease in humans. We also found a set of proteins whose expression levels may be specific of the atrophying process. These proteins constitute potential biomarkers witnessing the activation of muscle atrophy and/or potential therapeutic targets.

Skeletal muscle mass is reduced during many diseases or physiological situations (disuse, aging), which results in decreased strength and increased mortality. Muscle mass is mainly controlled by the ubiquitin-proteasome system (UPS), involving hundreds of ubiquitinating enzymes (E2s and E3s) that target their dedicated substrates for subsequent degradation. We recently demonstrated that MuRF1, an E3 ubiquitin ligase known to bind to sarcomeric proteins (telethonin, α-actin, myosins) during catabolic situations, interacts with 5 different E2 enzymes and that these E2-MuRF1 couples are able to target telethonin, a small sarcomeric protein, for degradation. Amongst the E2s interacting with MuRF1, E2E1 was peculiar as the presence of the substrate was necessary for optimal MuRF1-E2E1 interaction. In this work, we focused on the putative role of E2E1 during skeletal muscle atrophy. We found that E2E1 expression was restricted to type I and type IIA muscle fibers and was not detectable in type IIB fibers. This strongly suggests that E2E1 targets are fiber-specific and may be strongly linked to the contractile and metabolic properties of the skeletal muscle. However, E2E1 knockdown was not sufficient for preserving the protein content in C2C12 myotubes subjected to a catabolic state (dexamethasone treatment), suggesting that E2E1 is not involved in the development of muscle atrophy. By contrast, E2E1 knockdown aggravated the atrophying process in both catabolic C2C12 myotubes and the Tibialis anterior muscle of mice, suggesting that E2E1 has a protective effect on muscle mass.

Une perte de masse musculaire complique de nombreuses situations pathologiques telles le cancer ou l'insuffisance rénale. Elle contribue au syndrome de fragilité et est associée à un risque accru de décès. Les études conduites sur des modèles animaux ont révélées le rôle prépondérant de l'activation des systèmes de protéolyse musculaire et notamment du système ubiquitine protéasome (UPS). Cependant, les études conduites chez l'homme restent rares. L'objectif de ce travail était donc d'identifier les mécanismes communs qui caractérisent les muscles en situation d'atrophie chez l'homme. Des biopsies musculaires chirurgicales ont été réalisées au cours d'interventions programmées chez des patient ayant un cancer bronchique (LC, n = 7), hémodialysés chroniques (HD, n = 7) ou indemnes de pathologies (CT, n = 7). Une quantification de l'expression de gènes des systèmes protéolytiques ubiquitine protéasome, de l'autophagie et de l'apoptose a été réalisée par qRT-PCR. Une analyse globale du protéome musculaire soluble a été conduite par Shot-Gun en spectrométrie de masse. Nous avons retrouvé une augmentation de l'expression de plusieurs acteurs de l'UPS et de l'autophagie chez les patients LC et HD, et de marqueurs d'apoptose chez les patients HD. La spectrométrie de masse nous a permis d'identifier plus de 1700 protéines. Une ACP a révélé 3 profils protéomiques différents correspondant aux trois groupes de patients. Une O-PLS-DA a été utilisée pour créer un modèle permettant de distinguer les muscles des patients malades de ceux des patients sains. Les protéines qui contribuaient le plus au modèle ont été retenues. Une analyse fonctionnelle a permis de déterminer que ces protéines étaient impliquées dans la protéolyse, le métabolisme des acides aminés, la glycolyse, la phosphorylation oxydative, la défense contre le stress oxydant, la contraction musculaire, la matrice extracellulaire et l'organisation du cytosquelette musculaire. Une analyse de réseau de co-expression a retrouvé des résultats concordant. Enfin, une étude de réseau d'interaction est en faveur d'une activation de la voie Wnt-bêtacaténine. Cette étude démontre chez l'homme, l'exécution d'un programme d'atrophie musculaire commun indépendamment de la pathologie causale. Il existe une activation de l'UPS mais aussi de l'autophagie. Elle s'accompagne de modification métaboliques caractérisée par une augmentation de la glycolyse et une dysfonction mitochondriale. Une augmentation du stress oxydant participerait à l'induction de ces changements. L'activation continue de la voie Wnt serait à l'origine de fibrose musculaire. Les mécanismes en jeu sont autant de pistes pour la mise au point de traitements de l'atrophie musculaire.

As a result of an imbalance between protein synthesis and proteolysis, important protein losses occur during various physiological and pathological situations. Skeletal muscle mass is largely controlled by the ubiquitin proteasome system and thus by ubiquitinating enzymes. This proteolytic system is highly regulated and controls precisely the activity of numerous cellular proteins that are first tagged by a polyubiquitin chain on an internal lysine. Ubiquitination is based on the sequential action of the E1-activating enzyme, an E2 ubiquitin-conjugating enzyme (38 members) and an E3 ligase (> 600 members). The E1 enzyme activates the ubiquitin that it is transferred to an E2. E3 ligases recruit and bind specific substrates. MuRF1, a muscle-speci_c E3, is the only E3 ligase known to target contractile proteins (α-actin,myosins) for degradation during catabolic situations. MuRF1 is therefore a putative target for preventing muscle wasting. However, RING E3s like MuRF1 highly depend on ubiquitin conjugating enzymes E2s for ubiquitin chain formation. Our main objective was then to identify the E2s working in concert with MuRF1 to target myofibrillar proteins in atrophying skeletal muscles. We focused on 12 E2 enzymes that are expressed in the skeletal muscle or in atrophying skeletal muscles. Classical biochemical approach such as pull-down did not allow identifying any MuRF1 interacting partner among these E2s, suggesting that E2-MuRF1 interactions are weak and/or transient, consistent with previous studies reporting that E2-E3 interactions range from moderate to weak. We demonstrated that only sensitive and complementary interactomic approaches (Surface Plasmon Resonance, Yeast three-Hybrid and split-GFP) allowed the identi_cation of MuRF1 E2 partners. We identified five E2 enzymes that physically interacted with MuRF1. We showed that these E2s functionally interacted with MuRF1 since, in contrast with the non-interacting E2D2, their co-expression in HEK293T cells with MuRF1 led to increased MuRF1 substrate degradation. In conclusion,we report the first MuRF1-E2s network. This may prove valuable for deciphering the precise mechanisms involved in the muscle-atrophying program and for proposing new therapeutically

Abstract Background Muscle wasting is observed in the course of many diseases and also during physiological conditions (disuse, ageing). Skeletal muscle mass is largely controlled by the ubiquitin‐proteasome system and thus by the ubiquitinating enzymes (E2s and E3s) that target substrates for subsequent degradation. MuRF1 is the only E3 ubiquitin ligase known to target contractile proteins (α‐actin, myosins) during catabolic situations. However, MuRF1 depends on E2 ubiquitin‐conjugating enzymes for ubiquitin chain formation on the substrates. MuRF1‐E2 couples are therefore putative targets for preventing muscle wasting. Methods We focused on 14 E2 enzymes that are either expressed in skeletal muscle or up‐regulated during atrophying conditions. In this work, we demonstrated that only highly sensitive and complementary interactomic approaches (surface plasmon resonance, yeast three‐hybrid, and split green fluorescent protein) allowed the identification of MuRF1 E2 partners. Results Five E2 enzymes physically interacted with MuRF1, namely, E2E1, E2G1, E2J1, E2J2, and E2L3. Moreover, we demonstrated that MuRF1‐E2E1 and MuRF1‐E2J1 interactions are facilitated by telethonin, a newly identified MuRF1 substrate. We next showed that the five identified E2s functionally interacted with MuRF1 since, in contrast to the non‐interacting E2D2, their co‐expression in HEK293T cells with MuRF1 led to increased telethonin degradation. Finally, we showed that telethonin governed the affinity between MuRF1 and E2E1 or E2J1. Conclusions We report here the first MuRF1‐E2s network, which may prove valuable for deciphering the precise mechanisms involved in the atrophying muscle programme and for proposing new therapeutical approaches.

BackgroundSkeletal muscle protein loss is an adaptive response to various patho-physiological situations, and the ubiquitin proteasome system (UPS) is responsible for the degradation of the bulk of muscle proteins. The role of E2 ubiquitin-conjugating enzymes is still poorly understood in skeletal muscle.MethodsWe screened for E2s expression levels in C2C12 myotubes submitted to the catabolic glucocorticoid dexamethasone (Dex).ResultsOne micromolar Dex induced an accumulation of proteasome substrates (polyUb conjugates) and an overexpression of the muscle-specific E3 ligase MuRF1 and of six E2 enzymes, UBE2A, UBE2B, UBE2D1, UBE2D2, UBE2G1, and UBE2J1. However, only MuRF1 and UBE2B were sensitive to mild catabolic conditions (0.16M Dex). UBE2B knockdown induced a sharp decrease of total (-18%) and K48 (-28%) Ub conjugates, that is, proteasome substrates, indicating an important role of UBE2B in the overall protein breakdown in catabolic myotubes.ConclusionsInterestingly, these results indicate an important role of UBE2B on muscle protein homeostasis during catabolic conditions.

s of the 9th International Conference on Cachexia, Sarcopenia and Muscle Wasting, Berlin, Germany, 10–11 December 2016 (part 1) 1-01 New formulation based on anti-atrophic peptides and dendrimers for skeletal muscle atrophy treatment Johanna Ábrigo, Valeria Márquez-Miranda, Juan C. Rivera, Ingrid Araya-Durán, Javier Aravena, Nicolas Pacheco, Fernando D. González-Nilo & Claudio Cabello-Verrugio Laboratory of Biology and Molecular Physiopathology, Universidad Andres Bello, CBIB, Universidad Andrés Bello, IMII, Santiago, Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile, Fundación Fraunhofer Chile Research, SantiagoChile Background: Loss of muscle strength and myofibrillar proteins are key features in skeletal muscle atrophy by disuse. One of the main mechanisms involved is the over-activation of ubiquitin–proteasome pathway (UPP). Angiotensin-(1-7) [Ang-(1-7)], a vasoactive peptide with anti-atrophic activity in skeletal muscle, is rapidly degraded in vivo and inefficient as treatment therapy. Many peptide-delivery strategies have been studied, including direct injection or administration using osmotic pumps. Dendrimers are promising vehicles for the protection and transport of numerous bioactive molecules, being hydroxyl poly(amidoamine) (PAMAM-OH) suggested as an safe drug carriers without toxic effects during in vitro and in vivo applications. Aim: The aim of the study is to evaluate the effect of Ang(1-7)/PAMAM-OH dendrimer complex intraperitoneally (IP) administered in skeletal muscle atrophy induced by disuse. Methods: C57/BL10J mice were IP injected with vehicle (PBS), Ang-(1-7), PAMAM-OH, and Ang-(1-7)/PAMAM-OH, and 24 h after, immobilized in the lower hindlimb for 24 h or 14 days. Gastrocnemius muscle was extracted, and maximal isometric strength was measured by electrophysiological analyses. Histological analyses were made by hematoxylin eosin stain. Muscle fiber cross-sectional area (CSA) was determined estimating the minimal Feret’s diameter of cryosections stained with Wheat Germ Agglutinin (WGA). Myosin Heavy Chain (MHC) levels were determined by western blot. Atrogin-1 and MuRF-1 were evaluated by RT-qPCR. Results: IP administration of Ang-(1-7)/PAMAM-OH complex, but not Ang-(1-7) alone, avoided the decrease of muscle strength, diminution of fiber diameter and decrease of MHC protein levels in the skeletal muscle, induced by disuse. Ang-(1-7)/PAMAM-OH also prevented the increment of atrogin-1 and MuRF-1 expression. Conclusion: Ang-(1-7) IP administrated as complex with PAMAM-OH dendrimer avoided the atrophic effects in skeletal muscle induced by disuse. Ang-(1-7)/PAMAM-OH complex can be an efficient method in therapy for treatment of skeletal muscle atrophy. Funding: AFM #16670; FONDECYT #1120380, 1161646; IMII #P09-016-F; UNAB DI-741-15/N; PhD Scholarship CONICYT; F.G.N.; FCR-CSB 09CEII-6991, ACT1107, and RED CYTED 214RT0482; CINV. 1-02 Endotoxin-induced autophagy dependent on Beclin1/Bcl2 complex is decreased by Angiotensin(1-7) in skeletal muscle Juan Carlos Rivera, Johanna Abrigo, Mario Chiong, Michael Bader, Robson A. Santos, Enrique Brandan, María José Acuña & Claudio Cabello-Verrugio Laboratorio de Biología y Fisiopatología Molecular, Universidad Andrés Bello, IMII, ACCDiS, Universidad de Chile, Santiago, Chile, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany, National Institute in Science and Technology in Nanobiopharmaceutics, UFMG, Belo HorizonteBrazil, CARE, Department of Cell and Molecular Biology, Universidad Católica, Santiago, Chile Background and Aim: Autophagy is a key mechanism in endotoxin-induced cachexia by lipopolysaccharide (LPS). The vasoactive peptide of non-classical axis of renin angiotensin system, Angiotensin-(1-7) [Ang-(1-7)] and Mas receptor have anti-atrophic effects in cachexia, however, is still unknown the effect on autophagy and mechanism in cachectic skeletal muscle. Methods: C57BL/6 J (WT) or KO Mas receptor (KO) mice were treated with LPS in absence or presence of Ang-(1-7), and we evaluated autophagic proteins. C2C12 culture cells exposed to LPS and Ang-(1-7), and we analyze autophagic and MAPK proteins, and autophagosome formation. Results: Our results showed that Ang-(1-7) decreased the increment of LPS-induced LC3II/LC3I ratio protein levels in ABSTRACTS © 2016 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society of Sarcopenia, Cachexia and Wasting Disorders Journal of Cachexia, Sarcopenia and Muscle 2016; 7: 626–662 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12164 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. diaphragm muscle compared to control of WT mice. In KO Mas mice, Ang-(1-7) lost this effect. In culture cells exposed to LPS, Ang-(1-7) produced a reduction on the autophagy flux, LC3II/LC3I ratio and the amount of autophagosome. Interestingly, LPS increased autophagy by the disruption of the Beclin1/Bcl2 complex, while Ang-(1-7) restored its formation. Finally, Ang-(1-7) reduced LPS-activated p38MAPK and JNK, two signalling pathways involved in the separation of the Beclin1/Bcl2 complex in presence of LPS. Conclusion: We suggest that Ang-(1-7) is a new regulator of autophagy in endotoxin-induced cachexia by a mechanism dependent on MAPK and Beclin1/Bcl2 complex. Funding: Association-Francaise Contre Les Myopathies AFM #16670; FONDECYT #1161646, 1120380; IMII #P09-016-F; UNAB DI-741-15/N. Conicyt Scholarship #21141242 and #21161353. 1-03 Identification of E2 enzymes involved in MuRF1dependent skeletal muscle atrophy Cécile Polge, Agnès Claustre, Christiane Deval, Daniel Béchet, Lydie Combaret, Didier Attaix & Daniel Taillandier Unité de Nutrition Humaine (UMR 1019 INRA/Clermont Université), CRNH Auvergne 63000 Clermont-FerrandFrance Background and aims: The Ubiquitin Proteasome System (UPS) is the major actor of muscle wasting during various physio-pathological situations. This system involves an enzymatic cascade E1, E2, and E3. The targeting specificity of the UPS relies on the capacity of the system to first recognize (E3s) and then label (E2s) the proteins to be degraded generally with a polyubiquitin chain. E2–E3 interactions are therefore crucial as they determine the fate of the substrates. In the past 15 years, numerous experiments have contributed to depict an incomplete picture of the mechanisms responsible for myofibrillar proteolysis. This includes the discovery of muscle-specific E3 ligases (e.g. MuRF1) and the identification of the signaling pathways involved. Our main objective was to identify the E2-MuRF1 couples involved in the targeting of myofibrillar proteins in atrophying muscles. Methods: We focused on 14 E2 enzymes that are either abundant in skeletal muscle and/or up-regulated in atrophying conditions. We used control fa-C2C12 myotubes (expressing flag-actin) treated or not with dexamethasone (Dex, 1μM) and muscles from hindlimb suspensed rats to determine the expression levels of these enzymes. The MuRF1 cognate E2 enzymes were identified by knockdown and interactomic approaches. Results: Dex treatment increased mRNA levels of UBE2A, UBE2B, UBE2D1, UBE2D2, UBE2G1, and UBE2J1. UBE2A did not interact with MuRF1 and was not involved in contractile protein degradation. We next demonstrated that UBE2B was involved in α-actin and myosin heavy-chain destabilization in fa-C2C12 myotubes. However, this process was restricted to the cytoplasmic fraction and was MuRF1-independent. By contrast with previous assumptions, we found that UBE2D2 is not the MuRF1 partner for α-actin degradation in cellulo and is probably not involved in muscle wasting. Conclusions: UBE2B is the first E2 involved in contractile protein targeting, this action being presumably downstream of MuRF1 action. 1-04 Repetitive pulmonary inflammation in emphysematous mice induces sustained muscle wasting due to impaired muscle mass recovery Judith JM Ceelen, Annemie MWJ Schols, Stefan J van Hoof, Chiel C de Theije, Frank Verhaegen & Ramon CJ Langen Maastricht University Medical Center, Department of Respiratory Medicine, MaastrichtThe Netherlands, Department of Radiation Oncology (MaastRO) Background and aims: Exacerbations in COPD are often accompanied by pulmonary inflammation, and associated with increased prevalence of weight loss and muscle wasting. Emphysema-associated muscle atrophy may result from the cumulative effects of acute muscle mass loss during disease exacerbations, and subsequent impaired muscle regrowth. The aim of this study was to test whether muscle mass recovery following muscle atrophy induced by pulmonary inflammation is impaired in emphysematous mice and culminates in sustained muscle wasting. Methods: Emphysema was induced by 3 weekly intra-tracheal (IT) elastase instillations. Subsequently, 3 weekly boluses of ITLPS were administered to mimic a repetitive pulmonary inflammation-driven disease exacerbation. Using micro conebeam CT-scans, emphysema was verified, and muscle mass changes were monitored and correlated to muscle strength. At 2 and 3 days following the first IT-LPS administration and 7 days after the third IT-LPS bolus, skeletal muscle was collected for analyses. Results: Irrespective of emphysema, muscle weight and strength was reduced 48 h after the first bolus IT-LPS and recovered thereafter. mRNA and protein levels of genes of the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP) were upregulated 48 h following IT-LPS. In contrast, mTOR signaling was reduced 48 h post-induction of pulmonary inflammation in control and emphysematous mice. Importantly, muscle strength recovery following subsequent IT-LPS challenges was impaired in emphysematous mice, resulting in a sustained decre

The Ubiquitin Proteasome System (UPS) is mainly responsible for the increased protein breakdown observed in muscle wasting. The E3 ligase MuRF1 is so far the only enzyme known to direct the main contractile proteins for degradation (i.e. troponin I, myosin heavy chains and actin). However, MuRF1 does not possess any catalytic activity and thus depends on the presence of a dedicated E2 for catalyzing the covalent binding of polyubiquitin (polyUb) chains on the substrates. The E2 enzymes belonging to the UBE2D family are commonly used for in vitro ubiquitination assays but no experimental data suggesting their physiological role as bona fide MuRF1-interacting E2 enzymes are available. In this work, we first found that the mRNA levels of critical E3 enzymes implicated in the atrophying program (MuRF1, MAFbx, Nedd4 and to a lesser extent Mdm2) are tightly and rapidly controlled during the atrophy (up regulation) and recovery (down regulation) phases in the soleus muscle from hindlimb suspended rats. By contrast, E3 ligases (Ozz, ASB2β and E4b) implicated in other processes (muscle development or regeneration) poorly responded to atrophy and recovery. UBE2B, an E2 enzyme systematically up regulated in various catabolic situations, was controlled at the mRNA levels like the E3s implicated in the atrophying process. By contrast, UBE2D2 was progressively repressed during atrophy and recovery, which makes it a poor candidate for a role during muscle atrophy. In addition, UBE2D2 did not exhibit any affinity with MuRF1 using either yeast two-hybrid or Surface Plasmon Resonance (SPR) approaches. Finally, UBE2D2 was unable to promote the degradation of the MuRF1 substrate α-actin in HEK293T cells, suggesting that no functional interaction exists between these enzymes within a cellular context. Altogether, our data strongly suggest that UBE2D2 is not the cognate ubiquitinating enzyme for MuRF1 and that peculiar properties of UBE2D enzymes may have biased in vitro ubiquitination assays.

Acute Kidney Injury (AKI) is frequently encountered in hospitalized patients where it is associated with increased mortality and morbidity notably affecting muscle wasting. Increased protein degradation has been shown to be the main actor of AKI-induced muscle atrophy, but the proteolytic pathways involved are poorly known. The Ubiquitin Proteasome System (UPS) is almost systematically activated in various catabolic situations, and the E3 ligases MuRF1 and MAFbx are generally up regulated in atrophying muscles. We hypothesized that the UPS may be one of the main actors in catabolic skeletal muscles from AKI animals. We used gentamicin-induced acute kidney disease (G-AKI) in rats fed a high protein diet to promote acidosis. We first addressed the impact of G-AKI in the development of mild catabolic conditions. We found that both muscle atrophy and UPS activation were induced with the development of G-AKI. In addition, the phasic muscles were more sensitive to 7-days G-AKI (−11 to −17%, P<0.05) than the antigravity soleus muscle (−11%, NS), indicating a differential impact of AKI in the musculature. We observed an increased expression of the muscle-specific E3 ligases MuRF1 and MAFbx in phasic muscles that was highly correlated to the G-AKI severity (R2=0.64, P<0.01 and R2=0.71, P<0.005 respectively). Conversely, we observed no variation in the expression of three other E3 ligases (Nedd4, Trim32 and Fbxo30/MUSA1). Altogether, our data indicate that MuRF1 and MAFbx are sensitive markers and potential targets to prevent muscle atrophy during G-AKI.

Le systeme Ubiquitine Proteasome (UPS) est le principal acteur du controle de la masse musculaire au cours d’une situation catabolique. Les proteines a degrader sont marquees par une chaine d’ubiquitine impliquant une cascade enzymatique E1, E2, E3. Les combinaisons entre enzymes E2 (≥ 35) et E3 (≥ 600) permettent de virtuellement cibler n’importe quelle proteine de l’organisme. Les E3 reconnaissent les proteines a degrader mais ce sont les E2 qui portent generalement l’activite catalytique1. L’enzyme E3 MuRF1 est specifique du muscle squelettique et cible les proteines myofibrillaires majeures (actine, myosines, etc.) mais ce sont les couples E2-MuRF1 qui definissent le devenir des substrats. Les couples E2-MuRF1 representent donc une cible potentielle pour l’elaboration de strategies visant a reduire la perte de muscle squelettique, mais les enzymes E2 interagissant avec MuRF1 sont totalement inconnues. Notre objectif principal est donc d’identifier les E2 responsables de l’atrophie musculaire au cours de differentes situations cataboliques. Nos travaux se sont focalises sur 13 enzymes E2 abondantes dans le muscle squelettique et nous avons identifie 6 enzymes E2s (UBE2A, B, D1, D2, G1 et J1) dont les niveaux d’expression sont augmentes dans des myotubes en culture traites avec un agent catabolique, la dexamethasone (Dex, 1 μM). Nous avons ensuite utilise des approches de pulldown, de cribles double et triple hybride et de resonance plasmonique de surface (SPR) et nous avons demontre que les interactions E2-MuRF1 sont transitoires, labiles et que la presence d’un substrat est necessaire pour une interaction optimale. De facon interessante, nous avons demontre que UBE2D2 n’est pas un partenaire de MuRF1 bien que cette enzyme soit couramment utilisee pour des tests in vitro d’ubiquitination, ce qui implique que nous avons identifie les premiers couples E2-E3 potentiellement impliques dans le ciblage des proteines contractiles du muscle squelettique. La suite de nos travaux permettra d’identifier les proteines contractiles degradees par ces couples E2- MuRF1 et, a terme, d’envisager de nouvelles strategies permettant de limiter la perte de muscle squelettique au cours d’une situation catabolique

As a result of an imbalance between protein synthesis and/or proteolysis, important protein losses occur during various physiological and pathological situations. The ubiquitin proteasome system (UPS) is recognized as one of the major actor for controlling muscle mass. This proteolytic system controls precisely the activity of numerous cellular proteins that are first tagged by a polyubiquitin chain on an internal lysine. Ubiquitination is based on the sequential action of an E1-activating enzyme, an E2 ubiquitin-conjugating enzyme and an E3 ligase. The E1 ubiquitin-activating enzyme activates the ubiquitin that it is transferred to an E2. E3 ligases recruit and bind specific substrates. However, E2s are generally the main determinants that select the lysine for building ubiquitin chains, and thereby directly control the fate of the substrate. A given E2 can work with different E3s so that combinations between E2s (tens) and E3 ligases (hundreds) allow the targeting of virtually all cellular proteins. MuRF1 (Muscle Ring Finger 1) is one of the two E3 enzymes involved in muscle atrophy process during various catabolic situations. MuRF1 targets major myofibrillar proteins (troponin I, actin, myosin heavy chains, regulatory myosin light chains 1 and 2) for breakdown by the 26S proteasome. Elaborating new strategies to prevent muscle wasting requires information about the precise mechanisms of contractile protein degradation, including the recognition and the