I. M. Konstantinova

Role of proteasomes in cellular regulation.

The 26S proteasome is the key enzyme of the ubiquitin-dependent pathway of protein degradation. This energy-dependent nanomachine is composed of a 20S catalytic core and associated regulatory complexes. The eukaryotic 20S proteasomes demonstrate besides several kinds of peptidase activities, the endoribonuclease, protein-chaperone and DNA-helicase activities. Ubiquitin-proteasome pathway controls the levels of the key regulatory proteins in the cell and thus is essential for life and is involved in regulation of crucial cellular processes. Proteasome population in the cell is structurally and functionally heterogeneous. These complexes are subjected to tightly organized regulation, particularly, to a variety of posttranslational modifications. In this review we will summarize the current state of knowledge regarding proteasome participation in the control of cell cycle, apoptosis, differentiation, modulation of immune responses, reprogramming of these particles during these processes, their heterogeneity and involvement in the main levels of gene expression.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.

Changes in composition and activities of 26S proteasomes under the action of doxorubicin—apoptosis inductor of erythroleukemic K562 cells

Changes in the subunit composition, phosphorylation of the subunits, and regulation of the activities of 26S proteasomes in proliferating cells undergoing programmed cell death have not been studied so far. Moreover, there are no reports on phosphorylation of proteasome subunits both in normal and in neoplastic cells during apoptosis. The data of the present study show for the first time that apoptosis inductor doxorubicin regulates subunit composition, enzymatic activities, and phosphorylation state of 26S proteasomes in neoplastic (proerythroleukemic K562) cells or, in other words, induces reprogramming of proteasome population. Furthermore, the phosphorylation state of proteasomes is found to be the mechanism controlling specificity of proteasomal proteolytic and endoribonuclease activities.

Studies of poly(A+-RNA in mouse hepatoma and cortisone-stimulated rat liver

SummaryThe content of poly(A)-containing RNA in subcellular fractions has been investigated both in cortisone-treated rat liver and experimental hepatoma cells. The fractions included nuclei, cytoplasm, mitochondria, free and membrane-bound polyribosomes.1)In both cases of genome activation (cortisone induction and hepatoma cells) an increase in poly(A) content of all subcellular fractions except free polyribosomes was observed.2)Cortisone was found to induce elongation of poly(A) segments detected in both nuclei and cytoplasm.3)An increase in the poly(A) block size also was found to be stimulated in nuclei and cytoplasm of hepatoma cells.4)The observed elongation in poly(A) length occurred against the background of an increase of the population of poly(A)-RNAs.

PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response

Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5′ ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.

Regulation of the Specificity of the 26S Proteasome Endoribonuclease Activity in K562 Cells under the Action of Differentiation and Apoptosis Inducers

The specificity of the 26S proteasome endoribonuclease activity in proerythroleukemic K562 cells has been shown to change under the effects of inducers of erythroid differentiation inducers led to specific stimulation of RNase activity for certain mRNAs and to reduction of proteasome RNase activity for other mRNAs. The studied enzymatic activity was shown to be specifically and selectively dependent on phosphorylation of the 26S proteasome subunits, as well as on Mg and Ca ions. It was shown that the specificity of the proteasome RNase activity is regulated during differentiation and apoptosis. Selective regulation of the proteasome via the activities of different nuclease centers was suggested. This regulation may be accomplished through changes in the phosphorylation state of the proteasome subunits as well as by cation homeostasis.

The specific endoribonuclease activity of small nuclear and cytoplasmic α-RNPs

For the first time small nuclear ribonucleoprotein particles (α‐RNP) tightly bound to chromatin as well as cytoplasmic α‐RNP are shown to possess strong and regulated endonuclease activity specific for mRNAs and hnRNAs. The enzymatic nature of this activity is confirmed, and the optimal conditions detected. This RNase activity is controlled by the action of a differentiating stimulus, dimethylsulfoxide, in human K562 cells. Small α‐RNP involvement in the coordinated control of stability of pre‐messenger RNA and messenger RNA molecules is suggested.

Reprogramming of nuclear proteasomes under apoptosis induction in K562 cells II. Effect of antitumor drug doxorubicin

The induction of apoptosis in K562 cells by doxorbuicin was used as a model for studying changes of the subunit composition, phosphorylation state, and enzymatic activities of nuclear proteasomes undergoing programmed cell death. The proteasomes isolated from nuclei of the control and induced K562 cells have been shown to differ in their subunit composition, as well as in the phosphorylation state of subunits at threonine and tyrosine residues. Changes of the trypsin-and chymotrypsin-like, as well as endoribonuclease, activities of proteasomes under the doxorubicin action were revealed. After the induction of apoptosis in K562 cells by doxorubicin, we observed a modification of the RNase activity-associated proteasome subunits zeta/α5 and iota/α6. These results argue in favor of changes of proteasomal subunit composition, enzymatic activities, and the phosphorylation state, i.e., of the reprogramming of nuclear proteasome population, after the induction of apoptosis in K562 cells.

Reprogramming of Nuclear Proteasomes under Apoptosis Induction in K562 Cells I. Effect of Glutathione-depleting Agent Diethylmaleate

In the present work, changes in the subunit composition, phosphorylation state, and enzymatic activities of 26S proteasomes undergoing programmed cell death were studied. Apoptosis in proerythroleukemic K562 cells was induced by the glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and apoptotic K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. As well, the trypsin-and chymotrypsin-like activities of nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) were found to change under DEM action in K562 cells. DEM treatment of K562 cells led to a modification of proteasomal zeta/α5 and iota/α6 subunits associated with RNase activity. The obtained results argue in favor of changes of proteasomal subunit composition, phosphorylation state, and enzymatic activities, i.e., indicate the so-called reprogramming of the nuclear proteasome population during induced apoptosis in K562 cells.