Irina A. Oussenko

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3′ end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3′ extensions of the majority of CCA-less tRNAs.

Effect of ON 01910.Na, an Anticancer Mitotic Inhibitor, on Cell-Cycle Progression Correlates with RanGAP1 Hyperphosphorylation

The benzyl styryl sulfone, ON 01910.Na, is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell lines. We examined the effect of ON 01910.Na on DNA damage-signaling molecules upstream of Cdc25C (Chk1, Chk2, and H2AX), as well as on Ran GTPase-activating protein 1 conjugated to small ubiquitin-related modifier 1 (RanGAP1·SUMO1), a mitosis coordinator. Prostate cancer, lymphoma, and leukemic cells were incubated with the drug for 4, 16, or 24 hours. Cell lysates were resolved on SDS-PAGE and analyzed by Western blot. Camptothecin and doxorubicin treatment caused activation/phosphorylation of DNA damage-responsive molecules by 4 hours, whereas ON 01910.Na did not do so. ON 01910.Na caused hyperphosphorylation of RanGAP1·SUMO1 within 4 hours that was sustained for more than 24 hours. Mild phosphorylation of Chk2 was observed only after 24-hour exposure, indicating that DNA damage response was not an initial effect of ON 01910.Na. MOLT-3 cells, synchronized by double-thymidine block, when released into a medium containing ON 01910.Na, accumulated mitotic cell number with a peak from 10 to 14 hours and remained near plateau for 20 hours, which corresponded with the time of RanGAP phosphorylation. ON 01910.Na had minimal effects on tubulin polymerization. These findings imply that ON 01910.Na neither induces DNA damage directly nor acts as a tubulin toxin. Its biological activity appears to rely on prolonged phosphorylation/hyperphosphorylation of RanGAP1·SUMO1. M-phase arrest and the consequent induction of apoptosis that follows could possibly be attributed to it. ON 01910.Na may act as an inhibitor of a RanGAP1·SUMO1 phosphatase or a stimulant of a new kinase. RanGAP1·SUMO1 appears to be a new target pathway for cancer chemotherapy.

Assay of Bacillus subtilis ribonucleases in vitro.

Significant progress has been made recently regarding the identification of the ribonucleases involved in RNA maturation and degradation in Bacillus subtilis. More than half of these enzymes have no ortholog in Escherichia coli. To confirm that the in vivo effects of mutations in genes encoding RNases are direct, it is often necessary to purify the enzymes and assay their activity in vitro. Development of such assays is also necessary for detailed biochemical analysis of enzyme properties. In this chapter, we describe the purification and assay of 12 RNases of B. subtilis thought to be involved in stable RNA maturation or RNA degradation.

Abstract 4595: RPL18A as putative target of rigosertib

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Rigosertib (ON 01910.Na) is clinical stage anti-mitotic cancer inhibitor that causes spindle abnormalities in neoplastic cells. Rigosertib inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously we reported rigosertib led to hyperphosphorylation of RanGAP1•SUMO1, regulator of RanGTP/RanGDP cycle, and hypothesized phosphatase inhibition [Cancer Res 2011; 71; 4968-76]. Using biotin-conjugated rigosertib and avidin-conjugated agarose beads, we sought rigosertib molecular target from HeLa cell lysates by pull-down assay. Long-linker 1910-biotin showed an IC50 for cytotoxicity similar to unmodified rigosertib and efficient avidin binding. Asynchronously grown HeLa cell lysates were incubated with designated ligands and avidin beads. Bound lysate proteins were recovered from beads, resolved on SDS-PAGE and stained. Specific protein band of ∼20 kDa was detected only with long-linker 1910-biotin. Identification of proteins from the gel was performed by LC-ESI-MS/MS. Four ribosomal proteins possessed Mascot score above cut-off level with RPL18A, score of 367, and other proteins scored 153 or less. Specific dose-dependent binding of long-linker 1910-biotin to RPL18A was confirmed by Western blot analysis in replicate pull-down experiments using HeLa or MOLT-3 cell lysates. Recombinant RPL18A possessed specific reactivity to 1910-biotin in the pull-down assay. Knock-down of RPL18A with siRNA causes apoptosis in cancer cell lines [Sudo et al, Genomics 2010; 95; 210-6; and our data]. We propose that nucleocytoplasmic transport of RPL18A is impaired when hyperphosphorylated RanGAP1•SUMO1 fails to provide RanGTP/GDP gradient for nuclear pore complex. Importin-9 (IPO-9) is a major nucleocytoplasmic transporter for RPL18A. Among other cargos of IPO-9 are HSP27 (important anti-apoptotic player), scaffold subunit A of PP2A (major mitotic phosphatase), core histones and ribosomal proteins. Considering the essential role of nucleocytoplasmic transport in general and probable consequences of impeded transport of HSP27, PP2A and/or core histones, that actually match those observed with rigosertib, we hypothesize that binding with RPL18A in the presence of hyperphosphorylated RanGAP1•SUMO1 interferes with normal function of IPO-9, resulting in a variety of malfunctions and apoptosis observed in rigosertib treated cancer cells. Citation Format: Irina Oussenko, Saikrishna Divakar, M. V. Ramana Reddy, James F. Holland, E. Premkumar Reddy, Takao Ohnuma. RPL18A as putative target of rigosertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2014-4595

Epigenomic landscape of the human pathogen Clostridium difficile

Abstract Clostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed the first comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ~300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.Clostridium difficile is a leading cause of health care–associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been explored. Here, we performed the first DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. Strikingly, we discovered a DNA methyltransferase with a well-defined specificity, highly conserved across our dataset and in all the ∼300 global C. difficile genomes we further examined. Inactivation of the methyltransferase negatively impacted sporulation, a key step in C. difficile transmission, consistently supported by multi-omics data and genetic experiments and a mouse infection model. Transcriptomic analysis also suggested that epigenetic regulation is associated with host colonization and biofilm formation. The epigenomic landscape also allowed an integrative analysis of multiple defense systems with respect to their roles in host defense and in regulating gene flux in C. difficile. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.

Abstract 694: Structure-function analysis of RPL18A, a putative binding target of rigosertib

Rigosertib (ON 01910.Na; RGS) is a clinical stage anticancer agent that causes spindle abnormalities and mitotic arrest in neoplastic cells. The drug inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously, we reported identification of RPL18A (L18A), a protein from the large ribosomal subunit, as a putative binding target of RGS [Proc. AACR 2014, #4595]. Knock-down of L18A with siRNA caused apoptosis in cancer cell lines. Role of L18A in the function of the ribosome is not known. Goal of this study was to conduct structure-function analysis of L18A and create deficiency mutant(s) for future experiments. Human L18A is predicted to adopt the following linear order of secondary structural elements - alpha helices (a) and beta strands (b) - when assembled into the ribosome - b1b2a1b3b4b5b6a2a3b7a4 (from www.rcsb.org/pdb/protein/Q02543). The predicted 3D structure of L18A suggests overall bilobal architecture with a flanking C-terminal tail. Packed in tandem, each lobe contains one alpha helix surrounded by a beta sheet formed by 4 (N-lobe) or 3 (C-lobe) beta strands. To map the potential binding site for RGS, we engineered multiple mutants of L18A including truncations at its N and C termini. Our approach involved successive removal of the structural elements and a few substitutions with alanine. Mutant proteins were expressed in E.coli as GST fusions, and derivative cell lysates were tested in pull-down assay with biotin-conjugated RGS (RGSbio) and avidin beads. Pulled-down protein-drug complexes were analyzed by Western blot with anti-GST antibody. At the C-terminus, abolishing a4 helix (C - a4) preserved RGSbio-binding activity comparable to wild type (WT) L18A. Further C-truncations resulted in partial (C - a3b7a4) or complete loss of activity (C - a2→a4; C - b6→a4 and C - b5→a4; latter represents N-lobe while missing entire C-lobe). Unlike N-lobe, independently expressed C-lobe had specific binding activity, albeit half that of WT. At the N-terminus, stepwise truncation of amino acids 6 through 12 in the first beta strand (b1) resulted in gradual reduction of specific binding activity. To verify that this was not due to structure misfolding we tested mutants with single Ala substitutions in b1 and obtained similar results. Such mutants will serve as RGS-non-binding controls in future studies. In the model, L18A packs in the ribosome in a way that its surface at the side of two alpha helices of both lobes is engaged in protein-RNA interactions. However, the outer surface of the two cross-braced beta sheets in both lobes is completely exposed to solvent and interestingly, there is a cavity formed at the junction of two lobes that appears to be deep enough to accommodate one molecule of RGS. Taken together, our data suggest that rigosertib approaches RPL18A at the exposed hinge region between the two lobes and may thus perturb ribosomal function in cancer cells. Citation Format: Irina A. Oussenko, Yogesh K. Gupta, Rodrigo Vasquez-Del Carpio, M.V. Ramana-Reddy, Aneel K. Aggarwal, E. Premkumar Reddy, James F. Holland, Takao Ohnuma. Structure-function analysis of RPL18A, a putative binding target of rigosertib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2015-694

Abstract 3797: Hyperphosphorylation and desumoylation of RanGAP1·SUMO1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Previous work from our laboratory showed that tumor cells exposed to an anticancer mitotic inhibitor, regosertib (ON 01910.Na), resulted in G2/M arrest and hyperphosphorylation of RanGAP1·SUMO1 (HRGS). These findings suggest that regosertib may act either as an inhibitor of a RanGAP1·SUMO1 phosphatase or stimulant of a new kinase (Cancer Res 2011; 71; 4968-76). We further studied the relationship between dephosphorylation and deSUMOylation of HRGS. In the present study we used DU145 prostate cancer cell line. After exposure to one microM regosertib for 24 h, cells were collected and subjected, in equal portions, to lysis in different lysis buffers composed of basic lysis buffer and varied in supplemented inhibitors. Basic lysis buffer contained 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton; 0.5% NP-40; 1 mM EGTA and 1 mM EDTA. Thus, prepared cell lysates were analyzed by Western blot for expression of phosphorylated and non-phosphorylated forms of RanGAP1, RanGAP1·SUMO1, PP1 and Cdc25C. Results: Roche protease inhibitor cocktail, PMSF (0.5 mM), NaF (1 mM) and Na3VO4 (1mM) as inhibitors added to the basic lysis buffer were unable to protect against loss of SUMO-group from RanGAP1·SUMO1 and, concomitantly, against de-phosphorylation of PP1 phosphatase at T-320. However, addition of N-ethylmaleimide (NEM, an inhibitor of deSUMOylation) as a single inhibitor supplement in the basic lysis buffer resulted in concentration-dependent (2.5 ∼ 40mM) inhibition of deSUMOylation of HRGS. Yet, even at lower NEM concentration RanGAP1, devoid of the SUMO-group, was still phosphorylated. This might be in correlation with our finding that even low concentration of NEM in the lysis buffer kept PP1 in phosphorylated state at T320 (which is inhibitory to PP1), preventing it from auto-dephosphorylation and re-activation. Also, neither regosertib (up to 100 microM) nor okadaic acid (OA, up to 1 microM), added to the basic lysis buffer as putative inhibitors, could prevent loss of SUMO group from HRGS and dephosphorylation of PP1 at T320. Regosertib in the lysis buffer did not prevent dephosphorylation of hyperphosphorylated Cdc25C (aka mitotic Cdc25C), while NEM and OA did (the latter implies that dephosphorylation was carried out by PP1 in the lysate). Conclusion: These data show that hyperphosphorylated RanGAP1·SUMO1 can be deSUMOylated, but remains phosphorylated. DeSUMOylation may be a prerequisite for RanGAP dephosphosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3797. doi:1538-7445.AM2012-3797

Abstract 2639: RanGAP1·SUMO1 hyperphosphorylation (RGSH) and mitotic arrest by ON 01910.Na, okadaic acid or tubulin agents

INTRODUCTION: RanGAP1·SUMO1 is phosphorylated before nuclear envelope breakdown when cells enter mitosis. Previous work from our laboratory showed that a new anticancer antimitotic agent, ON 01910.Na (abbreviated 1910), a benzyl styryl sulfone analog, induced RGSH in all four human tumor cell lines tested. RGSH was not observed in the presence of 1911, inactive analog of 1910. However, RGSH was observed in the presence of tubulin depolymerizing agent nocodazole. Tubulin polymerization assay revealed that, unlike nocodazole, neither 1910 nor 1911 had effects on tubulin polymerization in vitro. In this study, we examined whether other tubulin agents induce RGSH and whether known mitotic phosphatases are involved. METHODS and RESULTS: We used two cell lines, MOLT-3 ALL and DU145 prostate cancer cell lines. We examined RGSH in the presence of known tubulin toxins including paclitaxel, vinblastine, vincristine, colchicine and nocodazole at a concentration of 1 µM in 4 h and 24 h. Doxorubicin and camptothecin served as negative controls. Following treatment with drugs, cells were analyzed by Western blotting. Drug concentrations that inhibited cell growth by 50% (IC50) or 90% (IC90) were determined in 3-day drug exposure experiments. All tubulin agents in the study were capable of inducing RGSH after 4h and 24 h exposure in both cell lines. The drug-induced RGSH was inhibited by caffeine, an ATM and ATR kinase inhibitor, and roscovitine, a Cdk kinase inhibitor. Okadaic acid (OA), known primarily as PP1 and PP2A phosphatase inhibitor, was also found to be capable of RGSH, therefore making it plausible by analogy that 1910 is RanGAP1·SUMO1 phosphatase inhibitor. The concentrations of 1910, OA or tubulin agents sufficient to elicit RGSH in 24 h, also caused 50 to 90% growth inhibition, respectively. 1910, as well as all tubulin agents in the study, induced phosphorylation of the phosphatase Cdc25C (aka “mitotic” Cdc25C) and down-regulation of interphase form of Cdc25C. CONCLUSION: 1910 and selected tubulin agents caused RGSH at IC50 – IC90 concentrations. Similar to tubulin agents, 1910 produced Cdc25C phosphorylation and down-regulation of its interphase form. OA inhibition of PP1 phosphatase and activation of RGSH suggest that 1910 could also serve as a phosphatase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2011-2639

Abstract 2500: ON 01910. Na, a clinical stage anticancer mitotic inhibitor, produces prolonged hyperphosphorylation of RanGAP1•SUMO1 as a potential mechanism of G2/M arrest and apoptosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC INTRODUCTION: The benzyl styryl sulfone ON 01910. Na (abbreviated as 1910) is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell cultures. The compound is currently in Phase 1 and 2 trials. Available data show the drug produces three major abnormalities in tumor cell lines: (a) aberrant cell division, including irregular chromosomal segregation and cytokinesis; (b) G2/M arrest and apoptosis in many tumor cells; and (c) decreased expression of Cdc25C phosphatase. Early data suggested that 1910 was a PLK1 inhibitor. Subsequent studies did not confirm this, although 1910 was found to inhibit the PLK pathway. Precise mechanism of action continues to be investigated. METHODS and RESULTS: We assessed DNA damage checkpoints throughout the cell cycle and effects on signaling molecules upstream of Cdc25C following treatment of DU145 prostate cancer, Bel7404 hepatoma, U937 lymphoma or MOLT-3 ALL cells with 1910. Camptothecin (CPT) and doxorubicin (DOX) served as positive controls. Cell lysates after drug exposure for 4, 16 or 24 h were resolved on SDS-PAGE and analyzed by Western blot for Chk1, Chk2, ATM, RanGAP1•SUMO1 and their phosphorylated forms. CPT and DOX exposure resulted in activation/phosphorylation of DNA damage-responsive molecules Chk1, Chk2 and ATM by 4 h, whereas 1910 did not. However, hyperphosphorylation of RanGAP1•SUMO1 was observed within 4 h and sustained for more than 24 h during 1910 exposure. This was also seen with the anti-tubulin agent, nocodazole, but not with ON 01911, an inactive analog of 1910. Mild phosphorylation of Chk2 was observed only after 24 h exposure, suggesting that DNA damage response is not a primary effect of 1910. MOLT-3 cells, synchronized by double thymidine block, were released into medium supplemented with 1910, which resulted in peak accumulation of G2/M cells by 9-12 h. The G2/M cell fraction remained in plateau for more than 20 h. This timeframe correlated with the hyperphosphorylation of RanGAP1•SUMO1. Cleaved Lamin B, detected by 16 h and thereafter in 1910-containing release medium, confirmed an active apoptotic process during this period. MOLT-3 cells released into drug-free medium reached G2/M peak at 9-10 h and continued to cycle with synchronized transition into G1 at 15-16 h. RanGAP1•SUMO1 was not hyperphosphorylated and no cleaved Lamin B was detected. Tubulin polymerization assay revealed that 1910 and ON 01911 had little or no effect, whereas nocodazole inhibited the process. CONCLUSION: These findings show that 1910 is neither a direct DNA damage response inducer nor a tubulin toxin, and suggest that 1910 is an inhibitor of RanGAP1•SUMO1 phosphatase. Its mechanism of action appears to rely on prolonged hyperphosphorylation of RanGAP1•SUMO1, leading to G2/M arrest and induction of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2500.