[Design of cell line stable expressing proteasomal subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag based on HEK293 cells].

A stable cell line based on HEK293 cells that expresses proteasome subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag has been selected. This chimera was shown to be incorporated completely into proteolitic active proteasomes. The created cell line can be used for further fluorescent studies of proteasomes localization in the cell.

[Extracellular proteasomes purification methods and the evaluation of proteasomal peptidase activities].

In this paper, we present a comparative analysis of different methods of purification of proteasomes from the culture medium in which proerithroleukemia human K562 cells were grown. The results obtained allowed us to purify proteasomes from samples of conditioned cell culture medium and control the quality of the proteasome preparations at all stages of their separation. Extracellular proteasomes purified via different approaches possess all the three types of peptidase activity described for intracellular counterparts.

[Characterization of the extracellular proteasomes and its interacting proteins by iTRAQ mass spectrometry].

The analysis of the extracellular proteasomes by isobaric tagging for relative and absolute quantifications (iTRAQ) mass spectrometry has been carried out. Here we show a standard set of 26S proteasomal subunits in the composition of the extracellular proteasomes. Moreover, extracellular proteasomes have a number of PA200 activators, which, as previously thought, are localized in the cell nucleus. Posttranslational modifications (PTMs) of subunits of the extracellular proteasomes were revealed by iTRAQ mass spectrometry. For the first time we have identified several ubiquitination and acetylation sites on subunits alpha2 (K196), alpha4 (K189 and K234), alpha6 (K217), and Rpn6 (A2). We have revealed a large number of proteasome-interacting proteins that are involved in various cell processes, such as transcription, DNA repair, translation, cytoskeletal proteins and the proteins of the ubiquitin-proteasome system (UPS). Immunoblot analysis has confirmed the interactions between purified extracellular proteasomes and nine proteins which were randomly selected from the set of interacting proteins.

[The comparative analysis of extra- and intracellular proteasomes from K562 cell line].

The comparative analysis of peptidase activities of extra- and intracellular proteasomes was carried out. Here we have shown that excreted proteasomes exhibit higher chymotrypsin-type and lower tripsin-like peptidase activities that cytoplasmic particles. Posttranslational modifications (PTMs) of 20S proteasomal subunits were revealed by immunoblotting techniques. We have observed the difference in PTMs of associated with enzymatic activities subunits beta2, beta5 and beta5 of extracellular and cytoplasmic proteasomes. Proteasomal subunits alpha2, 4, 7 and beta7 also had a variety of PTMs. The phosphorylation level of excreted proteasomes was lower compared to that of the intracellular ones. This observation strongly suggests the involvement of this PTM in the regulation of proteasomes excretion from cells.

[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.

[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.

[EGF regulation of specific endoribonuclease activity of 26S proteasomes from A431 cells: a potential role of proteasomes in control of mRNA stability]. / Reguliatsiia éndoribonukleaznoi aktivnosti 26S-proteasom épidermal'nym faktorom rosta v kletkakh linii A431: vozmozhnoe uchastie proteasom v kontrole nad stabil'nost'iu RNK.

For the first time it has been shown that RNase activity is induced under the influence of EGF on epidermoid carcinoma cell line A431. Proteasomes from EGF-treated A431 cells destabilize the 3'-untranslated regions of non-muscle beta actin mRNA, creating a specific cleavage pattern. In addition, these particles have been shown to specifically cleave Alu-containing informational RNA. The enzymatic activity under study has been shown to be dependent on phosphorylation of proteasomal subunits and specifically and selectively regulated by Ca and Mg ions. Proteasome involvement in the coordinated control of stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of 26S proteasomes can constitute a link between EGF signaling pathways and RNA stability.

[Properties of endoribonuclease activity of proteasomes from K562 cells. II. Analysis of specific mRNA nucleolysis by proteasomes]. / Kharacteristiki éndoribonukleaznoi aktivnosti proteasom iz kletok linii K562. II. Analiz nukleoliza spetsificheskikh mRNK proteasomami.

The ability of 26S proteasomes from the human proerythroleukaemic cell line K562 to degrade high-molecular-weight cytoplasmic RNAs, particularly specific messenger RNA, has been detected. The addition of hemin to K562 cells in the culture media leads to redistribution of proteasomes and their migration mainly to the cytoplasm. The human wild type p53 gene mRNA was shown to be specifically nucleolized by proteasomes. These particles displayed endoribonuclease activity towards mRNA for Renilla sp. luciferase. Proteasomes also specifically degraded Alu-containing mRNAs. A supposition is made about the involvement of proteasomes in stability control of specific RNA groups.

[Properties of endoribonuclease activity of proteasomes from K562 cells. III. The specific endonuclease activity of ribonucleoprotein particles (alpha-RNP) tightly bound to chromatin and containing 20S proteosomes]. / Kharacteristiki éndoribonukleaznoi aktivnosti proteasom iz kletok linii K562. III. Spetsificheskaia éndonukleaznaia aktivnost' ribonukleoproteinovykh chastits (alpha-RNP), prochno sviazannykh s khromatinom i soderzhashchikh 20S prosomy.

The present work demonstrates the ability of 20S proteasome-containing, tightly bound to chromatin RNP-particles (alpha-RNP) to endonucleolyse specific messenger RNAs (in particular, human mRNA for p53 gene and mRNA for luciferase from Renilla sp.). The dependence of individual mRNA endonucleolysis by alpha-RNP particles on both the substrate and enzyme was found.

[Characteristics of endoribonuclease activity of proteasomes from K562 cells. I. Dependence of RNAase activity of proteasome and alph-RNP on conditions of endonucleolysis reaction]. / Kharacteristiki éndoribonukleaznoi aktivnosti proteasom iz kletok linii K562. I. Zavisimost' RNKaznoi aktivnosti proteasom i alpha-RNP ot uslovii reaktsii éndonukleoliza.

Proteosomes from human proerythroleukaemic cell line K562 are found to degrade high molecular weight cytoplasmic RNAs, particularly ribosomal and specific messenger RNA. This activity was observed to be endoribonucleotylic. The induction of differentiation by erythroid pathway in K562 cells invokes augmentation of endonuclease activity in proteasomes. The number of characteristics of this enzymatic activity was investigated. Specificity of endonuclease of these RNPs is shown to be Ca- and Mg-dependent. Dephosphorylation of protein subunits suppresses RNase activity of proteasomes. Endonuclease of proteasomes is thermolabile. The examined activity depends on the secondary structure of substrate RNA. Protein subunits are responsible for ribonuclease activity of proteasomes rather than for low molecular weight RNAs associated with the complex.

[Specific regulated endonuclease activity of small RNP, containing prosomes from the A431 cell line: possible mechanisms of regulating the stability of RNA by epidermal growth factor]. / Spetsificheskaia reguliruemaia éndoribonukleaznaia aktivnost' malykh RNP, soderzhashchikh prosomy, iz kletok linii A431: vozmozhnyi mekhanizm reguliatsii stabil'nosti RNK épidermal'nym faktorom rosta.

A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.