title: UBE2D2 is not involved in MuRF1-dependent muscle wasting during hindlimb suspension.
abstract: 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.
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Polge
authors.affiliation: Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP 87, 38702, La Tronche Cedex, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Koulmann
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Claustre
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Jarzaguet
authors.affiliation: Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP 87, 38702, La Tronche Cedex, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Serrurier
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Lydie
authors.initials: L
authors.lastname: Combaret
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Béchet
authors.affiliation: Département Environnements Opérationnels, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP 87, 38702, La Tronche Cedex, France; French Anti-Doping Agency, Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Bigard
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Attaix
authors.affiliation: Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche (UMR) 1019, Unité de Nutrition Humaine (UNH), Centre de Recherche en Nutrition Humaine (CNRH) Auvergne, Clermont-Ferrand, France; Clermont Université, Université d'Auvergne, UNH, BP 10448, Clermont-Ferrand, France. Electronic address: daniel.taillandier@clermont.inra.fr.
authors.forename: Daniel
authors.initials: D
authors.lastname: Taillandier
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D019417:Hindlimb Suspension / Q000009:adverse effects*; D006801:Humans; D008297:Male; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009133:Muscular Atrophy / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D000070584:Tripartite Motif Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D009124:Muscle Proteins; D000070584:Tripartite Motif Proteins; D044763:Ubiquitin-Conjugating Enzymes; C499086:Trim63 protein, rat; D044767:Ubiquitin-Protein Ligases
keywords: MuRF1; Muscle wasting; UBE2D2; Ubiquitin-proteasome system