New putative phenol oxidase in ascidian blood cells.

The phenol oxidase system is ancient and ubiquitously distributed in all living organisms. In various groups it serves for the biosynthesis of pigments and neurotransmitters (dopamine), defence reactions and tissue hardening. Ascidians belong to subphylum Tunicata, which is considered the closest living relative to Vertebrates. Two phenol oxidases previously described for ascidians are vertebrate-like and arthropod-like phenol oxidases. In our present study, we described a new ascidian protein, Tuphoxin, with putative phenol oxidase function, which bears no sequence similarity with two enzymes described previously. The closest related proteins to Tuphoxin are mollusc haemocyanins. Unlike haemocyanins, which are oxygen transporting plasma proteins, Tuphoxin is synthesised in ascidian blood cells and secreted in the extracellular matrix of the tunic-ascidian outer coverings. Single mature transcript coding for this phenol oxidase can give several protein products of different sizes. Thus limited proteolysis of the initial protein is suggested. A unique feature of Tuphoxins and their homologues among Tunicata is the presence of thrombospondin first type repeats (TSP1) domain in their sequence which is supposed to provide interaction with extracellular matrix. The finding of TSP1 in the structure of phenol oxidases is new and we consider this to be an innovation of Tunicata evolutionary lineage.

[Mass-spectrometric analysis of proteasomal subunits possessing endoribonuclease activity].

Proteasomes act as the main apparatus of non-lysosomal intracellular proteolysis and participate in the regulation of most important cellular processes. Despite considerable progress in the understanding of proteasome's functioning, some issues, in particular, RNase activity of these ribonucleoprotein complexes and its regulation remain scarcely explored. In this paper we found several proteins corresponding by electrophoretic mobility to subunits of the complex 20S proteasome to possess endoribonuclease activity with respect to both sense and antisense sequences of the c-myc mRNA 3'-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins revealed in the samples the presence of 20S proteasome subunits--αl (PSMA6), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3). A number of novel phosphorylation sites in subunits αl (PSMA6) and α7 (PSMA3), and the form of subunit α5 (PSMA5) with a deletion of N-terminal 20 amino acid residues detected. The observed differences of individual subunits in the possession endonuclease activity could be apparently explained by postranslational modifications of these proteins, in particular--by phosphorylation. It is shown that the specificity of the proteasomal RNase activity varies after dephosphorylation and also influenced by Ca and Mg cations. The conclusions made about the impact of the PTM status of proteasome subunits on the specificity of their RNase activity.

[Non-canonical activities of the proteasomes].

26S proteasome is a highly specialized evolutionarily conserved ribonucleoprotein machine to instantly restore order in the intracellular proteome. It is composed of the 20S core particle and attached there to regulatory complexes. In addition to a comprehensively studied ubiquitin-proteasome pathway of protein degradation, proteasomes are involved (directly or indirectly) in most stages of the regulation of gene expression (at the levels of transcription, splicing, mRNA stability control, etc.). In the present review an attempt to systematize the recent literature on proteasomes' role in the regulation of transcription at all stages, including the modulation of the activity and stability of transcription factors, chromatin remodulation, elongation and termination of RNA synthesis is made. In addition, very interesting but controversial feature of the proteasome: their ability to bind and hydrolyze certain types of RNA, is observed. Obviously, universal role of proteasomes in gene regulation is determined by complicated composition of these protein complexes possessing a unique set of different enzymatic activities: ATPase/helicase, proteolytic and ribonuclease, which, depending on the case, can be used together or separately.

[Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

[Proteasomes and their role in transcriptional regulation].

26S proteasome is a multisubunit protein complex that plays a pivotal role in protein degradation. Proteasome's enzymatic activities--proteolytic, ATPase/helicase and RNase--can be used in regulation of multiple cellular processes. Recent studies confirm the major role of proteasomes in transcriptional regulation. Although various post-translational modifications of proteasome subunits have been described, relatively little is known about their functional effect on regulation of proteasome-dependent gene expression. In this article, we talk about the role of proteasomes in control of gene expression and different stages of transcription.

[The effect of red pigment on amyloidization of yeast proteins].

Amyloid bound thioflavine T fluorescence was studied in the lysates of yeast strains carrying mutations in genes ADE1 or ADE2 and accumulating red pigment, a result of polymerization of aminoimidazoleribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in the case of cells grown in media containing high concentration of adenine (100 mg/l) that blocks accumulation of red pigment. Blocks at first stages of purine biosynthesis de novo also impede red pigment and lead to the same effect on thioflavine fluorescence. At the same time induction of mutations in genes ADE1 or ADE2 in originally white prototrophic strains leads to considerable drop of fluorescence. A fraction of protein polymers was studied by agarose gel electrophoresis and this permitted to conclude that lowering of fluorescence intensity is indeed connected with the decrease of amyloid amount in cells accumulating red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with Thioflavine T. 2D-electrophoretic comparison of pellet proteins of red and white isogenic strains, followed by MALDI, allowed identification of 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins, involved in glucose metabolism, closely corresponding to prion-dependent proteins characterized in our previous work. We suppose that, binding amyloid fibrils, red pigment hinders formation of prion aggregates and also, blocking fibril contact with chaperones, impedes prion propagation.

[Comparison of crude lysate pellets of isogenic strains of yeast with different prion composition: identification of a set of prion-associated proteins].

A new approach: comparative analysis of proteins of the pellets of crude cell lysates of isogenic strains of Saccharomyces cerevisiae differing by their prion composition permitted to identify a large group of prion-associated proteins in yeast cells. 2D-electrophoresis followed by MALDI-analysis of a recipient [psi-] strain and of [PSI+] cytoductant led to identification of 35 proteins whose aggregation state responded to a shift of prion(s) content. Approximately half of these proteins belonged to functional groups of chaperones and enzyme involved in glucose metabolism. Notable were also proteins involved in translation, in oxidative stress response and in protein degradation. The data obtained are compared with the results of other groups who used other approaches to detecting proteins involved in prion aggregates.

[Recombinant proteasomal alpha-type subunits exhibit endoribonuclease activity].

26S proteasome is a multi-subunit protein complex that consists of the regulatory 19S and the catalytic 20S subcomplexes. The major cellular function of the proteasome is protein degradation. It has been found recently that the 20S particle, besides its proteolytic activity, also possesses endoribonuclease activity. The latter is mediated by two alpha-type subunits (alpha1 and alpha5). In this report we have analyzed the remaining alpha-type subunits for their ability to hydrolyze RNA. We found that all of the recombinant subunits tested exhibited endoribonuclease activity which depended 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 metabolism of RNA.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. II. Effect of anticancer drug doxorubicin].

The induction of apoptosis in K562 cells by doxorubicin (DR) was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed death. Here we have shown for the first time that proteasomes isolated from the nuclei of control and induced K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that trypsin- and chymotrypsin-like, and the endoribonuclease activities of nuclear 26S proteasomes are affected under influence of DR on K562 cells. Treatment of K562 cells with DR leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNase activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasomes population in undergoing apoptosis K562 cells which is manifested by changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. I. Effect of glutathione-depleting agent, diethylmaleate].

Here we have studied changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed cell death. Apoptosis in proerythroleukemic K562 cells was induced by glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and induces K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. We observed trypsin- and chymotrypsin-like activities on nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) to be changed under effect of DEM on K562 cells. Treatment of K562 cells with DEM leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNAse activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasome population in undergoing apoptosis K562 cells which is manifested by the changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

[Regulation of the 26S proteasomes' endoribonuclease activity specificity in k562 cells under effect of differentiation and apoptosis inductors].

The specificity of 26S proteasomes' endoribonuclease activity has been shown to be changed under effect of erythroid differentiation (hemin) and programmed cell death (diethylmaleate) inductors in proerythroleukemic K562 cells. Treatment of K562 cells with apoptosis and differentiation inductors leads to the specific stimulation of RNase activity towards certain mRNA and to reduction of proteasome RNase activity towards other mRNA. The enzymatic activity under study has been demonstrated to be specifically and selectively dependent on phosphorylation of 26S proteasome subunits as well as on Mg and Ca ions. The conclusion is drawn that the specificity of the proteasomes' RNAse activity is regulated during differentiation and apoptosis, and selective regulation of the activity of different nuclease centers is suggested, the mechanism involving changes in phosphorylation of proteasome subunits and cation homeostasis.

[Testosterone's role in regulating expression of genes of several proliferation factors].

This research work focuses on an important topic--the study of cause and effect links between partial androgen deficiency of ageing men (PADAM) and an increased expression of genes of a series of factors that make proliferate activity. The results of this research show that an increased expression of genes of several proliferation factors, and a decreased expression of the gene of the insulin receptor among men of older age groups are all connected to PADAM. The given changes are directed at compensation for testicular inadequacy, and are a particular expression of metabolic syndrome (X-syndrome); their effect can be inversed however by androgen-replacement therapy.

[Specificity of changes in proteasome properties in diethylmaleate-induced apoptosis of K562 cells].

The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.

[Phosphorylation of proteasomes and alpha-RNP from rat liver cells].

In eukaryotic cells the population of proteasomes is heterogeneous. Here we have shown that proteasomes from nuclei and cytoplasm of rat liver cells differ in their subunit patterns. The subunit pattern of alpha-RNP differs from that of proteasomes, however, alpha-RNP particles contain the number of 26S proteasome subunits. Moreover, the proteasomes contain subunits of alpha-RNP. We have shown for the first time that nuclear proteasomes and alpha-RNP are hyperphosphorylated on threonine residues. Differences in phosphorylation state of subunits of nuclear and cytoplasmic proteasomes and alpha-RNP on threonine and tyrosine residues have been revealed. A suggestion is put forward that hyperphosphorylation of subunits may determine nuclear localization of these complexes in rat liver cells. The results obtained suggest that a highly specialized system of protein kinases and phosphatases may be involved in the regulation of phosphorylation state of different populations of proteasomes and alpha-RNP in rat liver cells.

[Effect of EGF on the activity of nuclear and cytoplasmic 26S proteasomes in A431 cells].

It has been first shown that EGF regulates a proteolytic activity of proteasomes. Following a 15 min action with 100 ng/ml EGF, three types of peptidase activity of both cytoplasmic and nuclear proteasomes were induced in A431 cells, although, this effect on different populations of proteasomes was selective. EGF preferentially stimulates chymotrypsin-like activity of cytoplasmic proteasomes, and induces a similar increase of chymotrypsin-like, trypsin-like and peptydylglutamyl peptide hydrolase activities of nuclear particles. Tyrphostin, an inhibitor of tyrosine kinase activity of EGF receptor, prevents the EGF effect on both proteolytic and RNase activity of nuclear and cytoplasmic proteasomes. It is concluded that EGF may rapidly and selectively stimulate enzymatic activity of EGF receptor.

[Selective effect of epidermal growth factor on the endoribonuclease activity of different subpopulations of proteasomes from A431 cell line. Specificity of extracellular proteasome population]. / Selektivnoe vliianie épidermal'nogo faktora rosta na éndoribonukleaznuiu aktivnost' razlichnykh subpopuliatsii proteasom v kletkakh linii A431. Spetsifichnost' populiatsii proteasom, éksportiruemykh iz kletok.

For the first time, it has been shown that population of proteasomes is heterogeneous in their RNAse activity. EGF exerts selective effect on different subpopulations of proteasomes. The RNAse activity of cytoplasmic proteasomes is induced under the influence of EGF on epidermoid carcinoma cell line A431. However, the activity of proteasomes isolated from culture medium and of nuclear proteasomes is inhibited by EGF. The above enzymatic activity has been shown to be specifically and selectively dependent on phosphorylation of proteasomal subunits in different subpopulations of proteasomes. Proteasome involvement in the coordinated control of specific messenger RNA molecules stability is suggested, and one of the mechanisms of this control might be an export of specific subpopulation of proteasomes from the cell.

[Selective effect of inductors of apoptosis on the endoribonuclease activity of 26S proteasomes and alpha-RNP particles in K562 cells: possible involvement of 26S proteasomes and alpha-RNP in the regulation of RNA stability]. / Differentsial'naia reguliatsiia éndoribonukleaznoi aktivnosti 26S-proteasom i alpha-RNP-chastits pri deistvii induktorov apoptoza na kletki linii K562: vozmozhnoe uchastie alpha-RNP-chastits i proteasom v kontrole nad stabil'nost'iu RNK pri programmirovannoi kletochnoi gibeli.

It has been shown that endoribonuclease activity of alpha-RNP particles and 26S proteasomes are changed under the action of inductors of programmed cell death. Treatment of K562 cells with inductors of apoptosis--doxorubicin (adriamycin) and diethylmaleate--lead to a significant stimulation of RNAse activity of alpha-RNP and to reduction of proteasome RNase activity. The enzymatic activity under study has been shown to be specifically and selectively dependent on phosphorylation of subunits of alpha-RNP particles and 26S proteasomes. The characteristics of RNAse activity of different subpopulations of proteasomes differ. The specificity of a subpopulation of proteasomes exported from the cell has been demonstrated. Proteasome and alpha-RNP involvement in the coordinated control of stability of various specific messenger RNA molecules is suggested, and one of the mechanisms of this control might be the export of specific subpopulation of proteasomes from the cell.