Valentina A. Kulichkova,
Senior Scientist, Ph.D.

 Alexey G. Mittenberg,  
Senior Scientist, Ph.D.

Several important discoveries have been made in the laboratory in respect to the role of non-canonical activities of proteasomes and proteasome-like complexes in regulation of gene expression. First, small RNA-associated Particles (α-RNPs) have been identified and characterized. Further studies demonstrated that α-RNPs are tightly bound to chromatin. Moreover, they contain small RNAs, including Alu (SINE)-like ones. Sequencing of RNAs purified from rat liver α-RNP particle population has identified a novel type of small RNAs - U86 (GenBank). The composition and enzymatic properties of α-RNPs were found similar, but not identical, to proteasomes.

Original data have been obtained on participation of α-RNPs and proteasomes in the control of lifespan of mRNA molecules and participation of small antisense RNAs in hormonal control of gene expression. Also, the role of proteasomes in the programmed cell death has been investigated.

Second, a large volume of data has been obtained on the role and specificity of the endoribonuclease activity of 26S proteasomes. It has been found that 26S proteasomes preferably cleave AU-rich RNAs. Significantly, the AU-rich sequences are located in 3'-UTRs of various mRNAs coding for important transcription factors (e.g. p53 and c-myc).

Furthermore, this activity is regulated by extra-cellular stimuli (hemin, doxorubicin, Glucocorticoid Hormones, and Epidermal Growth Factor) via yet unknown mechanisms. In addition, specific proteasomal subunits possessing RNase activity were identified. Collectively, these results argue in favor of participation of these complexes (α-RNPs and 20S proteasomes) in differential regulation of lifespan of various mRNA molecules upon different extra-cellular signals.

Also, for the first time, 26S proteasomes were demonstrated to be excreted from cells under certain physiological conditions. Strikingly, an opposite process, i.e. import of proteasomes by living cells, has also been shown to take place. The proteasomes internalized by cells were shown to be enzymatically active, as they affected expression of apoptotic genes.

The main scientific focus of the Laboratory is to study various roles of proteasomes and α-RNP in regulation of gene expression. This includes several research objectives:

• Characterize the protein composition of α-RNPs;
• identify the spectrum of small non-coding RNAs associated with 26S proteasomes and their gene targets;
• investigate the mechanisms of cellular export/import of 26S proteasomes and their role for intercellular communication;
• investigate the mechanisms of 26S proteasome-dependent control of the lifespan of mRNA molecules.

The studies are carried out by using modern methods of biochemistry, molecular and cell biology, including electrophoresis of proteins and nucleic acids (both RNA and DNA) under native or denaturing conditions; two-dimensional electrophoresis of complex protein mixtures; Northern-, Southern-, and Western-blotting; PCR and RT-PCR; chromatography of proteins and nucleic acids (ion-exchange chromatography, gel-filtration), transcription in vitro, etc.

The work of the Laboratory is supported by grants from the Russian Foundation for Basic Research, Russian Federation Ministry of Science, Federal programs "Human genome", "Frontiers in genetics", "Frontiers in development of science and technology", and the European Commission (the 6^th framework program for support of cooperation with third countries). Also, the research is funded by The President of Russian Federation award, The Programs of the Ministry of Education & Science FASI (Contract No. P1389), The St. Petersburg Administration Scientific Award, the grant from St. Petersburg Scientific Center of RAS.

Several important discoveries in the field of gene regulation by post-translational covalent modifications have been made by the members of this group. Specifically, acetylation of tumor suppressor p53 was found to promote its activity in response to genotoxic stress by increasing the association between the former and histone acetyltransferase p300/CBP. Also, a novel covalent modification of p53, lysine methylation, exerted by a putative histone methyltransferase Set7/9 was identified. Importantly, lysine methylation of p53 was shown to facilitate the appearance of previously described modification, acetylation. Acetylation, in turn, prevents p53 poly-ubiquitinylation and its subsequent degradation by 26S proteasome.

It should be noted, that the proteolytic activity of 26S proteasome is itself subject to regulation by ubiquitinylation and phosphorylation induced by DNA damage.

In addition, several members of the 26S proteasome complex have been demonstrated to possess RNase activity, which is dynamically controlled by phosphorylation in a stimulus-specific manner. The mRNA stability of several important transcription factors, including p53 and c-myc, was found to be regulated by RNAse activity of the proteasome. This activity may be important for regulation of gene expression during genotoxic stress.

The major research goal of the group is to elucidate the role of post-translational modifications in regulation of transcriptional response during genotoxic stress. Accordingly, the group's research objectives are as follows:

• Study the role of lysine methylation, acetylation and ubiquitinylation of p53 and other factors in regulation of cell cycle progression and apoptosis in response to DNA damage;
• investigate the effects of phosphorylation of the 20S proteasomal components on transcription of pro-apoptotic genes upon DNA damage;
• elucidate the role of phosphorylation in regulation of RNase activity of several subunits of the 20S complex and their effect on stability of small non-coding micro-RNAs.

State-of-the-art technologies are being utilized to address these research aims. Specifically, in vitro arrays of 200 known nuclear kinases are currently being developed to facilitate the studies on the role of phosphorylation in regulation of DNA damage-controlled transcription. In addition, a wide variety modern techniques of cell biology, molecular biology, and biochemistry are used in the research including (but not limited to): 2D electrophoresis, mass-spectrometry, indirect immunofluorescent microscopy, FACs analysis, protein-protein interactions, western blotting, northern hybridization, RT-PCR, FPLC chromatography, transcription in vitro etc.

The research of the group is supported by grants from Molecular and Cellular Biology Program of RAS, Russian Foundation for Basic Research, Association for International Cancer Research, and National Institute of Health /NCI.

Several scientific collaborations have been established, including the University of Leicester, Leicester, UK, The Wolfson Institute at the University College of London, London, UK, The University of Florida, Gainesville, FL, USA.

Konstantinova I.M., Kulichkova V.A., Petukhova O.A., Pozdnyakov S.G., Lubchenkov M.A. 1994. Glucocorticoids specifically regulate expression of dispersed repeated sequences in liver cells. Detection of small ID-like antisense RNA. Mol. Biol. 28 (4):555-561. In Russian.

Konstantinova I.M., Petukhova O.A., Kulichkova V.A., Turoverova L.V., Volkova I.V., Ilkaeva, O.P., Kozhukharova, I.V., Ermolaeva Yu.B. 1995. A new class of RNA-containing RNP particles homologous to short dispersed DNA sequences. Mol. Biol. 29 (6): 761-771. In Russian.

Barlev N.A., Candau R., Wang L., Darpino P., Silverman N., Berger S.L. 1995. Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein. Journal of Biological Chemistry. 270: 19337-19344.

Candau R., Moore P., Wang L., Barlev N.A., Ying C., Berger S.L. 1996. Identification of functionally conserved human homologues of the yeast adaptors ADA2 and GCN5. Molecular and Cellular Biology. 16: 593-602.

Konstantinova I.M., Kulichkova V.A., Petukhova O.A., Turoverova L.V., Volkova I.V., Ignatova T.N., Kozhukharova I.V., Ermolaeva Yu.B., Mittenberg A.G., Gauze L.N. 1996. A new class of small ribonucleoprotein complexes (RNP) in human K-562 line cells. DAN. 350 (2): 268-271.

Wang L., Mizzen C, Ying C, Candau R., Barlev N., Brownell J., Allis С.D., Berger S.L. 1997. Histone acetyltransferase activity is conserved between yeast and human GCN5, and is required for complementation of growth and transcriptional activation. Molecular and Cellular Biology 7: 519-527.

Konstantinova I.M., Wetzker P., Petukhova O.A., Chesnokov I.N., Volkova I.V., Turoverova L.V., Ermolaeva Yu.B., Gauze L.N. 1997. A new class of small RNP (α-RNP) containing antisense RNA in K-562 line cells. III. DNA-binding nuclear α-RNP activity. Russ. J. Dev. Biol. (Ontogenez). 28 (2): 171-177.

Konstantinova I.M., Gauze L.N. 1997. The α-ribonucleoprotein: the world in a single particle. J. of journals. 1: 61-64.

Barlev N.A., Poltoratsky V., Owen-Hughes T., Ying C., Liu L., Carter T., Workman J., Berger S.L. 1998. Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the Ku/DNA-PKcs complex. Mol. Cell. Biol. 18: 1349-1358.

Jensen D.E., Proctor M., Marquis S.Т., Gardner H.P., Ha S.I., Chodosh L.A., Ishov A.M., Tommerup N., Vissing H., Sekido Y., Minna J., Borodovsky A., Schultz D.C, Wilkinson K.D., Maul G.G., Barlev N., Berger S.L., Prendergast G.C., Rauscher F.J. 3rd. 1998. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene. 16: 1097-1112.

Kulichkova V.A., Volkova I.V., Turoverova L.V., Evteeva I.N., Ermolaeva Yu.B., Mittenberg A.G., Urusova M.E., Galkin V.E., Gauze L.N., Konstantinova I.M. 1999. Specific DNA-binding and endoribonuclease activities of RNP firmly bound to chromatin, Mol. Biol. 33 (5): 764-772. In Russian.

Pleskach V.A., Ilkaeva O.R., Filatova N.A., Kozhukharova I.V., Turoverova L.V., Atochina O.V., Konstantinova I.M. 1999. Export and absorption of α-RNP with immunomodulating activity in cell cultures, DAN. 365 (6): 700-703.

Konstantinova I.M., Kulichkova V.A., Evteeva I.N., Mittenberg A.G., Volkova I.V., Ermolaeva J.B., Gause L.N. 1999. The specific endoribonuclease activity of small nuclear and cytoplasmic α-RNP. FEBS Lett. 462: 407-410.

Yan Y., Barlev N.A., Haley R.H., Berger S.L., Marmorstein R. 2000. Crystal structure of yeast esal suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases. Molecular Cell. 6: 1195-1205.

Barlev N.A., Liu L., Chehab N.H., Mansfield K., Harris K.G., Halazonetis T.D., Berger S.L. 2001. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Molecular Cell. 8: 1243-1254.

Mittenberg A.G., Kulichkova V.A., Medvedeva N.D., Penniyainen V.A., Gauze L.N., Konstantinova I.M. 2002. Characteristics of endoribonuclease activity of proteasomes from the K 562 line cells. I. Dependence of RNase activity of proteasomes and α-RNP on endonuclease reaction conditions. Tsitologiya. 44 (2): 181-187. In Russian.

Mittenberg A.G., Kulichkova V.A., Medvedeva N.D., Volkova, I.V., Ermolaeva Yu.B., Konstantinova I.M. 2002. Characteristics of endoribonuclease activity of proteasomes from the K 562 line cells. II Analysis of nucleolysis of specific mRNAs by proteasomes. Tsitologiya. 44 (4): 357-363. In Russian.

Mittenberg A.G., Kulichkova V.A., Gauze L.N., Konstantinova I.M. 2002. 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 proteasomes. Tsitologiya. 44 (4): 364-368. In Russian.

Gogolevskaya I.K., Makarova J.A., Gause L.N., Kulichkova V.A., Konstantinova I.M., Kramerov D.A. 2002. U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells. Gene. 292: 199-204.

Barlev N.A., Emelyanov A.V., Castagnino P., Zegerman P., Bannister A.J., Sepulveda M.A., Robert F., Tora L., Kouzarides T., Birshtein B.K., Berger S.L. 2003. A novel human Ada2 homologue functions with Gcn5 or Brg1 to coactivate transcription. Mol. Cell. Biol. 23: 6944-6957.

Goo Y.H., Sohn Y.C., Kim D.H., Kim S.W., Kang M.J., Jung D.J., Kwak E., Barlev N.A., Berger S.L., Chow V.T., Roeder R.G., Azorsa D.O., Meltzer P.S., Suh P.G., Song E.J., Lee K.J., Lee Y.C., Lee J.W. 2003. Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins. Molecular and Cellular Biology. 23: 140-149.

Evteeva I.N., Kulichkova V.A., Obukhova A.D., Mittenberg A.G., Volkova I.V., Ermolaeva Iu.B., Toktarova M.V., Teslenko L.V., Gauze L.N., Konstantinova I.M. 2003. EGF regulation of specific endoribonuclease activity of 26S proteasomes from A431 cells: a potential role of proteasomes in control of mRNA stability. Tsitologiya. 45 (5): 488-492, in Russian.

Toktarova M.V., Kulichkova V.A., Mittenberg A.G., Kozhukharova I.V., Volkova I.V., Ermolaeva Iu.B., Peshekhonov A.V., Ignatova T.N., Gauze L.N., Konstantinova I.M. 2004. 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. Tsitologiya. 46 (3): 283-290, in Russian.

Kulichkova V.A., Mittenberg A.G., Evteeva I.N., Toktarova M.V., Tsimokha A.S., Volkova I.V., Ermolaeva Yu.B., Gause L.N., Konstantinova I.M. 2004. Selective effect of epidermal growth factor on the endoribonuclease activity of different subpopulations of proteasomes from A431 cell line. Specificity of extracellular proteasome population. Tsitologiya. 46 (6): 525-530, in Russian.

Kulichkova V.A., Mittenberg A.G., Ermolaeva Yu.B., Volkova I.V., Kozhukharova I.V., Gause L.N., Konstantinova I.M. 2004. Specificity of the proteasome population secreted from cells into the culture medium. DAN. 399(5): 503-506, in Russian.

Chuikov S., Kurash Y., Wilson J.R., Xiao B., Justin N., Ivanov G., McKinney K., Tempst P., Prives C., Gamblin S., Barlev N., Reinberg D. 2004. Regulation of p53 activity through lysine methylation. Nature. 432: 353-360.

Tsimokha A.S., Vatazhok Yu.Ya., Vashukova E.S., Kulichkova V.A., Volkova I.V., Ermolaeva Yu.B., Mittenberg A.G., Evteeva I.N., Konstantinova I.M. 2005. Subunits of proteasomes and α-RNP from rat liver cells are phosphorylated at tyrosine and threonine. Tsitologiya. 47 (5): 436-441. In Russian.

Kulichkova V.A., Ermolaeva Yu.B., Mittenberg A.G., Volkova I.V., Tsimokha A.S., Evteeva I.N., Gauze L.N., Konstantinova I.M. 2005. Effect of EGF on activities of nuclear and cytoplasmic proteasomes in A431 cells. Tsitologiya. 47 (9): 774-779. In Russian.

Tsimokha A.S., Mittenberg A.G., Kulichkova V.A., Evteeva I.N., Vatazhok Yu.Ya., Moiseeva T.N., Ermolaeva Yu.B., Volkova I.V., Kozhukharova I.V., Gauze L.N., Konstantinova I.M. 2006. Specificity of changes of proteasomes during diethylmaleate-induced apoptosis in K562 cells. Tsitologiya. 48 (2): 133-144. In Russian.

Morgunkova A., Barlev N.A. 2006. Lysine methylation goes global. Cell Cycle. 5: 1308-1312.

Ivanov G.S., Ivanova T., Kurash J., Ivanov A., Chuikov S., Gizatullin F., Herrera-Medina E.M., Rauscher F., 3rd, Reinberg D., Barlev N.A. 2007. Methylation-Acetylation Interplay Activates p53 in Response to DNA Damage. Mol. Cell. Biol. 27: 6756-6769.

Mittenberg A.G., Pugacheva I.V., Moiseeva T.N., Tsimokha A.S., Kulichkova V.A., Gause L.N., Konstantinova I.M. 2007. Regulation of the Specificity of the 26S Proteasome Endoribonuclease Activity in K562 Cells under the Action of Differentiation and Apoptosis Inducers. Cell and Tissue Biology. 1 (2): 162-168.

Tsimokha A.S., Kulichkova V.A., Mittenberg A.G., Evteeva I.N., Kojukharova I.V., Ermolaeva J.B., Volkova I.V., Ignatova T.N., Gause L.N., Konstantinova I.M. 2007. Changes in composition and activities of 26S proteasomes under effect of diethylmaleate - inductor of apoptosis in erythroleukemic K562 cells. Cell Biol. Intl. 31: 338-348.

Tsimokha A.S., Mittenberg A.G., Kulichkova V.A., Vashukova E.S., Vatajok Yu.Ya., Moiseeva T.N., Evteeva I.N., Ermolaeva Yu.B., Gause L.N., Konstantinova I.M. 2007. Reprogramming of nuclear proteasome undergoing apoptosis in K562 cells. I. Effect of glutathione-depleting agent diethylmaleate. Cell and Tissue Biology. 1 (4): 334-342.

Tsimokha A.S., Mittenberg A.G., Evteeva I.N., Kulichkova V.A., Moiseeva T.N., Vatajok Yu.Ya., Vashukova E.S., Kojukharova I.V., Gause L.N., Konstantinova I.M. 2007. Reprogramming of nuclear proteasome undergoing apoptosis in K562 cells. II. Effect of anticancer drug doxorubicin. Cell and Tissue Biology. 1 (5): 404-412.

Myakishev M., Polesskaya O., Kulichkova V., Baranova A., Gause L., Konstantinova I. 2008. PCR-based detection of Pol III-transcribed transposons. Cell stress and chaperones. 13 (1): 111-116.

Tsimokha A.S., Vatajok Yu.Ya., Kulichkova V.A., Mittenberg A.G., Konstantinova I.M. 2008. Changes in content and proteolytic activities of proteasomes during apoptosis in K562 cells. Proceedings of Belarus NAS. 1: 112-118.

Mittenberg A.G., Moiseeva T.N., Barlev N.A. 2008. The role of proteasomes in transcription and their regulation by post-translational modifications. Frontiers in Bioscience. 13: 7184-7192.

Konstantinova I.M., Tsimokha A.S., Mittenberg A.G. 2008. Role of Proteasomes in Cellular Regulation. International Reviews of Cellular and Molecular Biology. 267: 59-124.

Moiseeva T.N., Semenova N.Y., Kuchina P.V., Mittenberg A.G. 2008. The endoribonuclease activity of proteasomal subunits and its regulation by post-translational modifications. Scholarly Letters of Kazan State University. p.67-69. In Russian.

Kulichkova V.A., Zaykova J.J., Tsimokha A.S., Kozhukharova I.V., Ermolaeva J.B., Evteeva I.N., Mittenberg A.G., Gauze L.N., Konstantinova I.M. 2008. Effect of exogeneous proteasomes on gene expression in cells through internalization. DAN. 423 (6): 464-468.

Kulichkova V.A., Fedorova O.A., Tsimokha A.S., Moiseeva T.N., Bottril A., Lezina L., Gauze L.N., Konstantinova I.M., Mittenberg A.G., Barlev N.A. 2010. 26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli. Cell Cycle. 9 (4): 840-849.

Moiseeva T.N., Mittenberg A.G., Barlev N.A. 2010. Proteasomes and their role in transcriptional regulation. Tsitologiya. 52 (3): 195-203.

Barlev N.A., Sayan B., Candi E., Okorokov A. 2010. The MicroRNA and p53 families join forces against cancer" Cell Death Differentiation. 17: 373-375.

Tsimokha A.S. 2010. Proteasomes: participation in cellular processes. Tsitologiya. 52 (4): 277-300.