Muscle atrophy prevails in numerous diseases (cancer cachexia, renal failure, infections, etc.), mainly results from elevated proteolysis, and is accelerated by bed rest. This largely contributes to increased health costs. Devising new strategies to prevent muscle wasting is a major clinical challenge. The ubiquitin proteasome system (UPS) degrades myofibrillar proteins, but the precise mechanisms responsible for actin breakdown are surprisingly poorly characterized. We report that chimeric flag-actin was destabilized and polyubiquitinylated in stably transfected C2C12 myotubes treated with the catabolic agent dexamethasone (1 μM) and that only proteasome inhibitors blocked its breakdown. Actin polyubiquitinylation was also detected in wild-type C2C12 myotubes and human muscle biopsies from control participants and patients with cancer. The muscle-specific E3 ubiquitin ligase MuRF1 is up-regulated in catabolic conditions and polyubiquitinylates components of the thick filament. We also demonstrate that recombinant GST-MuRF1 physically interacted and polyubiquitinylated actin in vitro and that MuRF1 is a critical component for actin breakdown, since MuRF1 siRNA stabilized flag-actin. These data identify unambiguously the abundant contractile protein actin as a target of the UPS in skeletal muscle both in vitro and in vivo, further supporting the need for new strategies blocking specifically the activation of this pathway in muscle wasting conditions.

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzymemediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified ␣2, ␣6, ␣7, ␤2, ␤3, ␤4, and ␤5 20 S proteasome subunits as potential substrates. Western blots using an anti-␤3 proteasome subunit antibody confirmed that high molecular weight forms of ␤3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.