Limiting cap-dependent translation increases 20S proteasomal degradation and protects the proteomic integrity in autophagy-deficient skeletal muscle

Postmitotic skeletal muscle critically depends on tightly regulated protein degradation to maintain proteomic stability. Impaired macroautophagy/autophagy-lysosomal or ubiquitin-proteasomal protein degradation causes the accumulation of damaged proteins, ultimately accelerating muscle dysfunction with age. While <i>in vitro</i> studies have demonstrated the complementary nature of these systems, their interplay at the organism levels remains poorly understood. Here, our study reveals novel insights into this complex relationship in autophagy-deficient skeletal muscle. We demonstrated that despite a compensatory increase in proteasome level in response to autophagy impairment, 26S proteasome activity was not proportionally enhanced in autophagy-deficient skeletal muscle. This functional deficit was partly attributed to reduced ATP levels to fuel the 26S proteasome. Remarkably, we found that activation of EIF4EBP1, a crucial inhibitor of cap-dependent translation, restored and even augmented proteasomal function through dual mechanisms. First, genetically activating EIF4EBP1 enhanced both ATP-dependent 26S proteasome and ATP-independent 20S proteasome activities, thereby expanding overall protein degradation capacity. Second, EIF4EBP1 activation caused muscle fiber transformation and increased mitochondrial biogenesis, thus replenishing ATP levels for 26S proteasome activation. Notably, the improved performance of the 20S proteasome in EIF4EBP1-activated skeletal muscle was attributed to an increased abundance of the immunoproteasome, a subtype specially adapted to function under oxidative stress conditions. This dual action of EIF4EBP1 activation preserved proteomic integrity in autophagy-deficient skeletal muscle. Our findings uncover a novel role of EIF4EBP1 in improving protein quality control, presenting a promising therapeutic strategy for autophagy-related muscular disorders and potentially other conditions characterized by proteostatic imbalance.<b>Abbreviations</b>: 3-MA: 3-methyladenine; ACAC/ACC: acetyl-Coenzyme A carboxylase; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; ATP: adenosine triphosphate; ATP5F1A/ATP5A: ATP synthase F1 subunit alpha; CKM-Cre: creatine kinase, muscle-Cre; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSK: cathepsin K; CTSL: cathepsin L; CUL3: cullin 3; EDL: extensor digitorum longus; EIF4E: eukaryotic translation initiation factor 4E; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EIF4F: eukaryotic translation initiation factor 4F complex; FBXO32/ATROGIN1/MAFbx: F-box protein 32; GFP: green fluorescent protein; IFNG/IFN-γ: interferon gamma; KEAP1: kelch-like ECH-associated protein 1; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; <i>Myl1/Mlc1f</i>-Cre: myosin, light polypeptide 1 (promoter driving Cre recombinase); mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NFE2L1/NRF1: nuclear factor, erythroid derived 2, like 1; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NFKB1/NFκB1: nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; OXPHOS: oxidative phosphorylation; PPARGC1A/PGC1α: peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; PSMB6: proteasome (prosome, macropain) subunit, beta type 6; PSMB7: proteasome (prosome, macropain) subunit, beta type 7; PSMB8: proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7); PSMB9: proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2); PSMB10: proteasome (prosome, macropain) subunit, beta type 10; PSME1: proteasome (prosome, macropain) activator subunit 1 (PA28 alpha); PSME2: proteasome (prosome, macropain) activator subunit 2 (PA28 beta); RBX1: ring-box 1; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1; STAT3: signal transducer and activator of transcription 3; TRIM63/MURF1: tripartite motif-containing 63; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system.