Caspase-3 Cleaves Specific 19 S Proteasome Subunits in Skeletal Muscle Stimulating Proteasome Activity

Abstract

With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically to increase the degradation of muscle proteins. Whether proteasome activity is also elevated in response to catabolic conditions is unknown. We find that caspase-3 increases proteasome activity in myotubes but not in myoblasts. This difference is related to the cleavage of specific 19 S proteasome subunits. In mouse muscle or myotubes, caspase-3 cleaves Rpt2 and Rpt6 increasing proteasome activity. In myoblasts, caspase-3 cleaves Rpt5 to decrease proteasome activity. To confirm the caspase-3 dependence, caspase-3 cleavage sites in Rpt2, Rpt6, or Rpt5 were mutated. This prevented the cleavage of these subunits by caspase-3 as well as the changes in proteasome activity. During differentiation of myoblasts to myotubes, there is an obligatory, transient increase in caspase-3 activity, accompanied by a corresponding increase in proteasome activity and cleavage of Rpt2 and Rpt6. Therefore, differentiation changes the proteasome type from sensitivity of Rpt5 to caspase-3 in myoblasts to sensitivity of Rpt2 and Rpt6 in myotubes. This novel mechanism identifies a feed-forward amplification that augments muscle proteolysis in catabolic conditions. Indeed, we found that in mice with a muscle wasting condition, chronic kidney disease, there was cleavage of subunits Rpt2 and Rpt6 and stimulation of proteasome activity.The ubiquitin-proteasome system (UPS) 4 is responsible for the degradation of most proteins in the cytoplasm and nuclei of cells (1, 2). It is also involved in regulating many cell functions including control of the cell cycle, antigen presentation, ion transport, and muscle mass (3-6). One event that regulates proteolysis in the UPS is the rate of ubiquitin conjugation to protein substrates. For example, ␤TCF, the activated E3 enzyme, triggers degradation of IB and ␤-catenin, whereas activation of Atrogin-1/MAFbx or MuRF1 in muscle is closely linked to increased protein degradation (7,8).Rates of protein degradation in the UPS could also be regulated through changes in the proteolytic activity of the proteasome. For example, treatment of lymphoid cells with ␥-interferon increases the expression of proteasome subunits, LMP2 and 7, and these subunits are incorporated into proteasomes stimulating the breakdown of proteins into peptides that are more suitable for class 1 antigen presentation (9 -11). Other evidence suggests that the activity of the UPS can be determined by variations in proteasome activity. In several conditions that are associated with the loss of muscle mass, proteasome subunits are expressed at a higher level but it is not proven that changes in subunit expression accelerate the rate of muscle proteolysis (12-18). Alternatively, proteasome activity can be suppressed in association with changes in subunits. Sun et al. (19) reported that caspase-3 activation in Jurkat T cells or cancer cells causes cleavage of specific subunits of the 19 S regulatory complex of the proteasome: Rpt5, Rpn10, and Rpn2...

Decreased miR-29 Suppresses Myogenesis in CKD

“…We showed that in muscle, IL-6 in synergy with the acute phase protein, serum amyloid A, stimulates activation of caspase-3 and the ubiquitin-proteasome system to increase protein degradation in muscle. 26,27 In addition, a mechanism involving myostatin has revealed how inflammation contributes to muscle wasting. 37 In that report, pharmacologic inhibition of myostatin not only reduced the high levels of circulating inflammatory cytokines including TNF␣ and IL-6 in CKD mice it also prevented muscle wasting and improved the differentiation of MPCs.…”

Assessment of skeletal muscle proteolysis and the regulatory response to nutrition and exercise

“…We have also demonstrated a reduction in post-exercise caspase-3 activation following mixed protein-carbohydrate consumption, relative to carbohydrate alone (24). In addition to cleaving myofibrils, caspase-3 may also serve to stimulate proteasome activity (25), thereby both providing substrate to the UPS and increasing UPS-mediated proteolysis.…”

Proteolysis in illness-associated skeletal muscle atrophy: from pathways to networks

“…Moreover insulin and IGF1 inhibit the production of the 14 kDa fragment through a PI3K-dependent mechanism (PI3K, phosphoinositide-3-kinase), suggesting that these hormones have a direct effect on CASP3 in addition to their inhibition of the UPS via FoxO as described below 141 . The nature of the activation of UPS proteolysis by CASP3 in catabolic conditions has been studied further; in addition to potentially cleaving and releasing substrates for the UPS, there is evidence that in muscle tissue CASP3 cleavage of two ATPase-containing subunits on the 19S proteasome regulatory particle [PSMC1 (RPT2) and PSMC5 (RPT6)] amplifies proteasome activity 142 .…”

“…In addition, caspase cleavage promotes atrophy by initiating myonuclear apoptosis. Caspases interact with other proteolytic systems including calpains as described above, and can also enhance proteasome activity 142 . Furthermore there is now evidence from studies in other cell types suggesting that caspase activity may have a role in actively directing a cell away from autophagy in favor of apoptosis through caspase cleavage of Beclin-1 145 .…”