MuRF1 partners with TRIM72 to impair insulin signalling in skeletal muscle cells

Abstract Muscle RING-finger protein 1 (MuRF1, gene name: TRIM63) is well known as a critical molecular regulator in skeletal muscle atrophy. Despite the identification of several substrates and interaction partners for MuRF1, the precise molecular mechanisms by which MuRF1 causes skeletal muscle atrophy remain unclear. To gain further insight into the underlying mechanism of skeletal muscle atrophy, we applied targeted biochemical approaches, and identified tripartite motif-containing protein 72 (TRIM72) as a novel MuRF1-interacting protein. Subsequent analysis using MuRF1 knockout and rescue experiments showed that TRIM72 protein abundance is dependent on the presence of MuRF1 protein. Furthermore, TRIM72 protein level was increased by dexamethasone treatment in C2C12 myotubes, alongside increased MuRF1 protein level. Dexamethasone decreases IRS1/Akt signalling, protein synthesis, and glucose uptake specifically in wild-type myotubes, but not in MuRF1 KO myotubes. Further analysis showed that overexpression of TRIM72 impairs IRS1/Akt signalling without the presence of MuRF1, indicating that MuRF1 induces a negative impact on insulin signalling through a plausible cooperation with TRIM72. Our findings provide novel non-degradative molecular roles of MuRF1 that link together skeletal muscle atrophy and impaired insulin responses. Highlights Identification of MuRF1 and TRIM72 interaction in skeletal muscle cells TRIM72 protein expression is dependent on the presence of MuRF1 protein Deletion of MuRF1 confers a protective effect against dexamethasone-induced impairment of IRS1/Akt signaling TRIM72 is sufficient to impair IRS1/Akt signaling Competing Interest Statement The authors have declared no competing interest. Abbreviations - Akt - protein kinase B - ANOVA - analysis of variance - ATP - Adenosine triphosphate - BSA - bovine serum albumin - Cas9 - CRISPR-associated protein - CO2 - carbon dioxide - Con - control - CRISPR - clustered regularly interspaced short palindromic repeats - Dex - Dexamethasone - DMEM - Dulbecco’s modified eagles’ medium - DNA - deoxyribonucleic acid - ER - endoplasmic reticulum. - FoxO - forkhead box - GAPDH - glyceraldehyde 3-phosphate dehydrogenase - GFP - green fluorescent protein - gRNA - Guide RNAs - GSK-3 - Glycogen synthase kinase-3 - h - hours - HCl - hydrochloric acid - HECT-type - homologous to the E6-AP C-terminus - IBR - Institute of Biomedical Research - IGF-1 - insulin like growth factor 1 - IRS1 - insulin receptor substrate 1 - kDa - kilodalton - KI - knock in - KO - knock out - mg - milligram - ml - millilitre - mmol - millimolar - MPB - muscle protein breakdown - MPS - muscle protein synthesis - mRNA - messenger ribonucleic acid - mTOR - mammalian target of rapamycin - mTORC1 - mammalian target of rapamycin complex 1 - mTORC2 - mammalian target of rapamycin complex 2 - MuRF1 - muscle-specific ring finger 1 - MuRF2 - muscle-specific ring finger 2 - MuRF3 - muscle-specific ring finger 3 - MyBP-C - myosin-binding protein C - MyLC1 - Myosin Light Chain 1 - MyLC2 - Myosin Light Chain 2 - O2 - oxygen - PBS - phosphate buffered saline - pH - potential of hydrogen - PI3K - phosphoinositide-3-kinase - PVDF - polyvinylidene fluoride - RhTRIM72 - Human recombinant TRIM72 - RING - Really Interesting New Genes - RNA - ribonucleic acid - SD - standard deviation of the mean - SDS - sodium dodecyl sulphate - SDS-PAGE - sodium dodecyl sulfate–polyacrylamide gel electrophoresis - SEM - standard error of the mean - Ser - serine - TBST - tris-buffered saline Tween-20 - Thr - threonine - TRIM25 - Tripartite Motif Containing 25 - TRIM32 - Tripartite Motif Containing 32 - TRIM63 - Tripartite Motif Containing 63 - TRIM72 - Tripartite Motif Containing 72 - UPS - ubiquitin proteasome system - WT - wild type