A. G. Mittenberg

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.

Prion-associated proteins in yeast: comparative analysis of isogenic [PSI(+)] and [psi(-)] strains.

A large group of prion‐associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two‐dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI+] and [psi−] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion‐containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion‐associated, but we cannot exclude the possibility that several proteins may propagate as prions. Copyright

The effect of red pigment on the amyloidization of yeast proteins

The intensity of amyloid‐bound thioflavine T fluorescence was studied in crude lysates of yeast strains carrying mutations in the ADE1 or ADE2 genes and accumulating the red pigment (a result of polymerization of aminoimidazoleribotide), and in white isogenic strains–either adenine prototrophs or carrying mutations at the first stages of purine biosynthesis. We found that the red pigment leads to a drop of amyloid content. This result, along with the data on separation of protein polymers of white and red strains in PAGE, suggests that the red pigment inhibits amyloid fibril formation. The differences in transmission of the thioflavine T fluorescence pattern by cytoduction and in blot‐hybridization of pellet proteins of red and white [PSI+] strains with Sup35p antibodies confirmed this conclusion. Purified red pigment treatment also led to a decrease of fluorescence intensity of thioflavine T bound to insulin fibrils and to yeast pellet protein aggregates from [PSI+] strains. This suggests red pigment interaction with amyloid fibrils. Comparison of pellet proteins from red and white isogenic strains separated by 2D‐electrophoresis followed by MALDI analysis has allowed us to identify 48 pigment‐dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, closely corresponding to prion‐dependent proteins that we characterized previously. Also present were some proteins involved in stress response and proteolysis. We suppose that the red pigment acts by blocking certain sites on amyloid fibrils that, in some cases, can lead in vivo to interfere with their contacts with chaperones and the generation of prion seeds. Copyright

Effect of red pigment on amyloidization of yeast

Amyloid-bound thioflavin T fluorescence was studied in lysates of yeast strains that carry mutations in the ADE1 or ADE2 genes and accumulate red pigment as a result of the polymerization of aminoimidazole ribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in cells grown in media with high concentrations of adenine (100 mg/l), which suppresses the accumulation of red pigment. Mutations that block the first stages of purine biosynthesis de novo also impede the accumulation of red pigment and produce the same effect on thioflavin fluorescence. Mutations in ADE1 or ADE2 genes in originally white prototrophic strains considerably suppress fluorescence. The fraction of protein polymers was studied by agarose gel electrophoresis, which permitted us to conclude that reduced fluorescence intensity was associated with decreased amyloid content in cells that accumulate red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with thioflavin T. A comparison of lysate pellet proteins from red and white isogenic strains separated by 2D electrophoresis followed by MALDI analysis allowed us to identify 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, which closely correspond to the prion-dependent proteins that we characterized previously. We suppose that the binding of red pigment with amyloid fibrils prevents the generation of prion aggregates and impedes prion propagation by blocking fibril contact with chaperones.

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.

Mass-spectrometric analysis of proteasome subunits exhibiting endoribonuclease activity

Proteasomes function as the main nonlysosomal machinery of intracellular proteolysis and are involved in the regulation of the majority of important cellular processes. Despite the considerable progress that has been made in understanding the functioning of proteasomes, some issues (in particular, the RNase activity of these ribonucleoprotein complexes and its regulation) remain poorly investigated. In this study, we found to several proteins with electrophoretic mobility that corresponds to that of 20S subunits of the core proteasome complex exhibit endoribonuclease activity with respect to the sense and antisense sequences of the c-myc mRNA 3′-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins showed that the samples contained 20S proteasome subunits—α1 (PSMA6), α5 (PSMA5), α6 (PSMA1), and α7 (PSMA3). A number of new phosphorylation sites of α1 (PSMA6) and α7 (PSMA3) subunits were found, and a form of α5 (PSMA5) subunit with a deletion of 20 N-terminal amino-acid residues was identified. The observed differences in the manifestation of endonuclease activity by individual subunits are apparently due to posttranslational modifications of these proteins (in particular, phosphorylation). It was shown that the specificity of RNase activity changes upon proteasome dephosphorylation and under the influence of Ca2+ and Mg2+ cations. It is concluded that posttranslational modifications of proteasome subunits affect the specificity of their RNase activity.

Recombinant proteasome alpha-type subunits exhibit endoribonuclease activity

Abstract26S proteasome is a multisubunit protein complex that consists of 19S regulatory and 20S catalytic subcomplexes. The primary proteasome cellular function is protein degradation. It has recently been found that, in addition to its proteolytic activities, the 20S particle also displays endoribonuclease activity mediated by two alpha-type subunits, α1 and α5. In this report, we have analyzed other alpha-type subunits for their ability to hydrolyze RNA. We have found that all of the recombinant subunits tested (α1, α2, α3, α4, α5, α7) exhibited endoribonuclease activity that depends on the origin of RNA and the presence of bivalent ions in the reaction. These results indicate that the endoribonuclease activity of proteasomes may play an important role in cellular RNA metabolism.