Importance of the ATP-Ubiquitin-Proteasome Pathway in the Degradation of Soluble and Myofibrillar Proteins in Rabbit Muscle Extracts

Abstract

Recent studies have suggested that activation of the ubiquitin-proteasome pathway is primarily responsible for the rapid loss of muscle proteins in various types of atrophy. The present studies were undertaken to test if different classes of muscle proteins are degraded by this pathway. In extracts of rabbit psoas muscle, the complete degradation of soluble proteins to amino acids was stimulated up to 6-fold by ATP. Peptide aldehyde inhibitors of the proteasome or the removal of proteasomes markedly inhibited only the ATP-dependent process. Addition of purified myosin, actin, troponin, or tropomyosin to these extracts showed that these proteins served as substrates for the ubiquitin-proteasome pathway. By contrast, degradation of myoglobin did not require ATP, proteasomes, or any known proteases in muscles.When myosin, actin, and troponin were added as actomyosin complexes or as intact myofibrils to these extracts, they were not hydrolyzed at a significant rate, probably because in these multicomponent complexes, these proteins are protected from degradation. Accordingly, actin (but not albumin or troponin) inhibited the degradation of 125 I-myosin, and actin was found to selectively inhibit ubiquitin conjugation to 125 I-myosin. Also, the presence of tropomyosin inhibited the degradation of 125 I-troponin. However, neither actin nor tropomyosin inhibited the degradation of 125 I-lysozyme or soluble muscle proteins. Thus, specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation, and the ratelimiting step in their degradation is probably their dissociation from the myofibril.

Regulation of Muscle Protein Degradation

“…Caspase-3 activation in muscle (Figure 2) is important because the Ub-P'some system does not degrade actomyosin, yet loss of actomyosin is characteristic of muscle atrophy ( Figure 1B) that occurs in catabolic conditions (1,28). The cleavage of actomyosin can be identified by the accumulation of the 14-kD actin band in muscle, a response that occurs in rats with chronic uremia or insulin deficiency (7).…”

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

“…However, these proteins were much more stable when associated with each other in the actomyosin complex or intact myofibrils. These findings raised the possibility that the ubiquitin-proteasome system may not by itself be able to degrade components of intact myofibrils, and constituent proteins have to be released by some mechanism to be substrates for degradation (Solomon and Goldberg, 1996). Consequently, several groups have presented evidence that a calpain (Tidball and Spencer, 2002) or caspase (Du et al, 2004) may initially cleave the myofibrillar components, thereby accelerating disassembly and degradation by the ubiquitin-proteasome system.…”

“…The degradation of myofibrillar components during atrophy is mediated by the ubiquitin-proteasome pathway (Solomon and Goldberg, 1996). Despite this rapid degradation of diverse muscle proteins during atrophy, two muscle-specific ubiquitin ligases (E3s), muscle RING-finger 1 (MuRF1) and atrogin-1/MAFbx, are markedly induced; and in knockout mice lacking either enzyme, the rapid loss of muscle mass upon denervation is significantly reduced (Bodine et al, 2001;Gomes et al, 2001).…”

X-chromosome linked inhibitor of apoptosis protein inhibits muscle proteolysis in insulin-deficient mice

“…13 We find that accelerated muscle protein degradation in streptozotocin (STZ)-induced insulin-deficient animals is because of activation of the ubiquitin-proteasome system; 13,14 however, this system cannot directly degrade myofibrillar proteins. 15 These proteins must first be cleaved by caspase-3 into constituent protein fragments to be degraded by the proteasome-dependent proteolysis system. The appearance of a 14-kDa actin fragment is a biomarker of myofibrillar protein breakdown.…”