Rapid Deubiquitination of Nucleosomal Histones in Human Tumor Cells Caused by Proteasome Inhibitors and Stress Response Inducers: Effects on Replication, Transcription, Translation, and the Cellular Stress Response

Abstract

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.