UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy

Abstract

Key points We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining. In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy. We demonstrated that UBR5 was epigenetically altered via DNA methylation during recovery from atrophy. We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy. UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation. Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast‐twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes. Abstract We aimed to investigate a novel and uncharacterized E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 vs. well characterized E3‐ligases, MuRF1/MAFbx, where, after atrophy evoked by continuous‐low‐frequency electrical‐stimulation in rats, MuRF1/MAFbx were both elevated, yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post TTX‐induced atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, whereas a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, as well as after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three‐dimensional bioengineered mouse muscle in vitro, and after chronic electrical stimulation‐induced hypertrophy in rats in vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload‐induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 single nucleotide polymorphisms) demonstrated that the A alleles of rs10505025 and rs4734621 single nucleotide polymorphisms in the UBR5 gene were strongly associated with larger cross‐sectional area of fast‐twitch muscle fibres and favoured strength/power vs. endurance/untrained phenotypes. Overall, we suggest that: (i) UBR5 comprises a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx; (ii) UBR5 is epigenetically regulated; and (iii) UBR5 is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.

Sarcopenia: Tilting the Balance of Protein Homeostasis

“…Thus, this supports the idea that UPP acts as a negative regulator of muscle hypertrophy under the influence of MAFbx and MuRF1, but promotes muscle hypertrophy with other proteins regulating UPP. It is worth noting that a recent study has found another E3 ligase, UBR5, with gene expression and protein levels increasing during periods of muscle recovery following muscle atrophy and seemed to be regulated inversely compared to MAFbx and MuRF1 during functional overloading in rats . Furthermore, UBR5 expression was found to be positively associated with muscle loading in untrained male subjects .…”

Identification and Characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase

“…In comparison, MuRF1 and MAFbx expression remain elevated for a longer time period in neurogenic muscle atrophy models, returning to baseline after 14 days. (9,12,16,22,35). Thus, we hypothesized that Fbxl22 could be involved in the early initiation of the muscle atrophy process.…”

“…Transfection of mouse skeletal muscle with plasmid DNA was performed in mice under isoflurane (2-4% inhalation) anesthesia as previously described (12,64). Briefly, after a 2hr pre-treatment with 0.4 units/µl of bovine placental hyaluronidase (Sigma) resuspended in sterile 0.9% saline, 20µg of plasmid DNA was injected into either the tibialis anterior (TA) or the lateral and medial gastrocnemius muscles (lateral gastrocnemius, LGA; medial gastrocnemius, MGA).…”

“…For RNAi experiments, the negative control RNAi plasmid was described previously (12,64) and encodes emerald green fluorescent protein (EmGFP) and a non-targeting pre-miRNA under bicistronic control of the cytomegalovirus (CMV) promoter in the pcDNA6.2GW/EmGFP-miR plasmid (Invitrogen, Carlsbad, CA). The Fbxl22 RNAi plasmid encodes EmGFP and an artificial pre-miRNA targeting the full length gene of mouse Fbxl22 under bicistronic control of the CMV promoter; it was generated by ligating the Mmi576126 oligonucleotide duplex (Invitrogen, Carlsbad, CA) into the pcDNA6.2GW/EmGFP-miR plasmid.…”

“…In skeletal muscle, only a few E3 ubiquitin ligases have been characterized and associated with muscle atrophy, including MuRF1 (muscle-specific RING finger 1) and MAFbx (muscle atrophy F-box; also known as Atrogin-1) (8,9), MUSA1 (muscle ubiquitin ligase of the SCF complex in atrophy-1) (10) and SMART (Specific of Muscle Atrophy and Regulated by Transcription) (11). Recently, we identified UBR5 as an E3 ubiquitin ligase that may play a role in muscle growth and hypertrophy (12). Considerably more E3 ubiquitin ligases are expressed in skeletal muscle, most of which remain to be explored in the context of muscle physiology and disease.…”