Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor.
Bottom Line: In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor.Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 microM), suggesting a requirement for new protein synthesis.These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia.
Affiliation: Department of Physiology and Biophysics, University of Campinas, UNICAMP, SP, Brazil 13083-970. M.J.Tisdale@aston.ac.ukShow MeSH
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Mentions: To determine whether PIF-induced protein catabolism was mediated through the ATP-ubiquitin-dependent pathway proteasome functional activity was determined by measuring ‘chymotrypsin-like’ enzyme activity, the major proteolytic activity of the β-subunits. Using the fluorogenic substrate succinyl LLVY-MCA an increase in enzyme activity was detectable at concentrations of PIF between 2 and 4 nM (Figure 1B). The effect on protein expression of proteasome subunits in the presence of PIF was determined by immunoblotting. Cellular supernatants of PIF-treated cells were Western blotted using MCP 231 antibody, a murine monoclonal to the 20S proteasome, which reacts with the six different α-type subunits. Three bands were detected at approximate molecular weight of 29 000, 32 000 and 35 000 (Figure 2AFigure 2
Affiliation: Department of Physiology and Biophysics, University of Campinas, UNICAMP, SP, Brazil 13083-970. M.J.Tisdale@aston.ac.uk