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.
Subject(s)
Caspase 3/metabolism , Gene Expression Regulation, Enzymologic , Muscle, Skeletal/enzymology , Proteasome Endopeptidase Complex/metabolism , RNA, Ribosomal/chemistry , Adenosine Triphosphate/chemistry , Animals , Caspases/metabolism , Humans , Jurkat Cells , Mice , Models, Biological , Mutation , Peptide Hydrolases/metabolism , Protein Structure, Tertiary , RNA, Ribosomal/metabolismABSTRACT
To develop improved methods of gene delivery, packaging DNA in chemical or viral vectors could increase electroporation-mediated transfection. To test this hypothesis, electroporation was applied to DU145 prostate cancer cells incubated with green fluorescent protein-encoded DNA plasmid either naked or packaged with cationic lipid (Lipofectin), polycationic peptide (salmon protamine) or retroviral vectors (Moloney murine leukemia viruses) and then assayed for gene expression and cell viability. Cationic lipid or electroporation alone each significantly increased transfection, but their combination was less effective. Addition of protamine peptide during electroporation was also less effective than electroporation alone. The combination of retroviral vectors and electroporation transfected fewer cells than retrovirus alone. We conclude that the combination of electroporation with chemical or viral vectors does not improve gene transfection in vitro.
