V. A. Kulichkova

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.

U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells.

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D, C, D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.

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.

Simultaneous EGFP and Tag Labeling of the β7 Subunit for Live Imaging and Affinity Purification of Functional Human Proteasomes

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the β7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the β7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to β7-EGFP-HTBH within the proteasome complex rather than to free protein. The β7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.

Proteomic analysis of affinity-purified extracellular proteasomes reveals exclusively 20S complexes

Proteasome-mediated proteolysis is important for many basic cellular processes. In addition to their functions in the cell, proteasomes have been found in physiological fluids of both healthy and diseased humans including cancer patients. Higher levels of these proteasomes are associated with higher cancer burden and stage. The etiology and functions of these proteasomes, referred to as circulating, plasmatic, or extracellular proteasomes (ex-PSs), are unclear. Here we show that human cancer cell lines, as well as human endometrium-derived mesenchymal stem cells (hMESCs), release proteasome complexes into culture medium (CM). To define ex-PS composition, we have affinity purified them from CM conditioned by human leukemia cell line K562. Using matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), we have identified core 20S proteasome subunits and a set of 15 proteasome-interacting proteins (PIPs), all previously described as exosome cargo proteins. Three of them, PPIase A, aldolase A, and transferrin, have never been reported as PIPs. The study provides compelling arguments that ex-PSs do not contain 19S or PA200 regulatory particles and are represented exclusively by the 20S complex.

Comparative analysis of extra- and intracellular proteasomes from K562 cells

In this manuscript we compared the biochemical properties of intracellular (cytoplasmic) and extracellular (secreted) proteasomes by their peptidase activities and the levels of phosphorylation of their subunits judged by relative mobility in two-dimensional gel electrophoresis and immunoblotting with phospho-specific antibodies. A comparative analysis of the peptidase activities of extra- and intracellular proteasomes showed that the excreted proteasomes exhibited higher chymotrypsin-like and lower trypsin-like peptidase activities than the cytoplasmic counterparts. The status of post-translational modifications, in particular phosphorylation, of 20S proteasomal subunits was also examined. We observed different levels of phosphorylation between the excreted and cytoplasmic proteasomes. According to the altered mobility in the two-dimensional electrophoresis of modified proteasome subunits combined with immunoblotting results using various phospho-specific antibodies, we concluded that subunits α2, α4, α7, and β7 were post-translationally modified. In addition, β2, β5, and α5 subunits, which possess enzymatic activities, were also differentially modified. Overall, the phosphorylation level of excreted proteasomes was lower than the intracellular ones. This observation indicates that the phosphorylation status of proteasomes may be important for their excretion from cells.