The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially purified from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-fluoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in vitro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.
Multicatalytic proteinase (MCP) is thought to play a central role in the processing and turnover of intracellular proteins in eukaryotic cells. Immunocytochemistry was used to determine the intracellular distribution of the MCP in the claw muscles of the land crab, Gecarcinus lateralis, and the claw and abdominal muscles of the American lobster, Homarus americanus. Cryosections were stained with an affinity-purified polyclonal antibody to lobster MCP that cross-reacted with the land crab enzyme. Two types of staining were observed: a diffuse cytoplasmic staining, and a dense aggregate staining primarily associated with invaginations of the cell membrane. The cytoplasmic staining appeared reticulated in favorable transverse sections due to a preferential localization of MCP to the intermyofibrillar space. The aggregate staining was associated with neither nuclei nor mitochondria, since stains specific for these organelles (Hoechst stain and nicotinamide adenine dinucleotide diaphorase histochemistry, respectively) did not colocalize with the aggregates. Trypsinlike peptidase activities of isolated microsomal and postmicrosomal fractions indicated that less than 1% of the total MCP was associated with the microsomal fraction. Immunoprecipitation of the same fractions confirmed the presence of MCP in the microsomes as well as in the cytosol. These results suggest that the MCP is primarily associated with cytoplasmic components; the aggregate staining may result from the association of the MCP with cellular membrane systems.
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