Renata Kowara

National Research Council Canada, Ottawa, Ontario, Canada

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Publications (16)30.73 Total impact

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    ABSTRACT: Dihydropyrimidinase-like 3 (DPYSL3) is believed to play a role in neuronal differentiation, axonal outgrowth and neuronal regeneration, as well as cytoskeleton organization. Recently we have shown that glutamate excitotoxicity and oxidative stress result in calpain-dependent cleavage of DPYSL3, and that NOS plays a role in this process [R. Kowara, Q. Chen, M. Milliken, B. Chakravarthy, Calpain-mediated truncation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H2O2 toxicity, J. Neurochem. 95 (2005) 466-474; R. Kowara, K.L. Moraleja, B. Chakravarthy, Involvement of nitric oxide synthase and ROS-mediated activation of L-type voltage-gated Ca(2+) channels in NMDA-induced DPYSL3 degradation, Brain Res. 1119 (2006) 40-49]. The present study investigates the involvement of PLA(2) signaling in NMDA-induced DPYSL3 degradation. Exposure of rat primary cortical neurons (PCN) to PLA(2) and COX-2 inhibitors significantly prevented NMDA-induced DPYSL3 degradation. Since the metabolic product of PLA(2) signaling, PGE(2), which augments toxic effect of NMDA, is known to stimulate cAMP, the effect of adenyl cyclase activator (forskolin plus IBMX) and inhibitor (MDL12,300) on NMDA-induced DPYSL3 degradation was tested. Our data indicate that the activation of adenyl cyclase contributes to NMDA-induced DPYSL3 degradation. Furthermore, cAMP-dependent protein kinase (PKA) inhibitor PKI (14-22) provided additional evidence of PKA involvement in NMDA-induced DPYSL3 degradation. In summary, the obtained data show the contribution of PLA(2) signaling to NMDA-induced calpain activation and subsequent degradation of synaptic protein DPYSL3.
    Neuroscience Letters 02/2008; 430(3):197-202. · 2.03 Impact Factor
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    ABSTRACT: Dihydropyrimidinase-like 3 (DPYSL3) and GAP43 are both involved in neurite outgrowth, a crucial process for the differentiation of neurons. The present study shows for the first time that DPYSL3 co-localizes with GAP43 in primary cortical neurons. Further co-immunoprecipitation and overlay assay showed the ability of both recombinant and endogenous DPYSL3 to bind GAP43, indicating a specific interaction between DPYSL3 and GAP43 in primary cortical neurons.
    Biochemical and Biophysical Research Communications 12/2007; 363(1):190-3. · 2.28 Impact Factor
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    ABSTRACT: Dihydropyrimidinase-like 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and possibly in neuronal regeneration. Recently, we have shown that in primary cortical neurons (PCN) NMDA and oxidative stress (H(2)O(2)) caused a calpain-dependent cleavage of DPYSL3 (62 kDa) resulting in the appearance of a lower molecular weight form (60 kDa) of DPYSL3. Our preliminary results had shown that antioxidants significantly reduced NMDA-induced DPYSL3 degradation, indicating involvement of ROS in calpain activation. The aim of this study was to investigate the possible involvement of NOS in NMDA-induced DPYSL3 degradation. We found that NOS inhibitor (L-NAME) significantly prevented NMDA-induced ROS formation, as well as intracellular Ca(2+) increase [Ca(2+)](i), DPYSL3 degradation and cell death. Further, exposure of PCN to NO donor (SNP) resulted in significant [Ca(2+)](i) increase, ROS generation and probable calpain-mediated DPYSL3 truncation. The NMDA- and oxidative stress (ROS)-induced DPYSL3 truncation was totally dependent on extracellular [Ca(2+)](i). While NMDA-induced DPYSL3 truncation was blocked by both NMDA receptor antagonist (MK801) [Kowara, R., Chen, Q., Milliken, M., Chakravarthy, B., 2005. Calpain-mediated degradation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H(2)O(2) toxicity. J. Neurochem. 95 (2), 466-474] and L-VGCC (nimodipine) inhibitors, H(2)O(2)-induced increase in [Ca(2+)](i), ROS generation and DPYSL3 truncation was blocked only by nimodipine. These results indicate that changes in Ca(2+) homeostasis resulting from ROS-dependent activation of L-VGCC are sufficient to induce probable calpain-mediated DPYSL3 truncation and demonstrate for the first time the role of ROS in the mechanism leading to glutamate-induced calpain activation and DPYSL3 protein degradation. The probable calpain-mediated DPYSL3 truncation may have significant impact on its interaction with actin and its assembly, and in turn on growth cone integrity.
    Brain Research 12/2006; 1119(1):40-9. · 2.88 Impact Factor
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    ABSTRACT: Dihydropyrimidinase-like protein 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and, possibly, neuronal regeneration. In primary cortical cultures, glutamate (NMDA) excitotoxicity and oxidative stress (H2O2) caused the cleavage of DPYSL3, resulting in the appearance of a doublet of 62 kDa and 60 kDa. Pre-treatment of cell cultures with calpain inhibitors, but not caspase 3 inhibitor, before exposure to NMDA or H2O2 completely blocked the appearance of the doublet, suggesting calpain-mediated truncation. Furthermore, in vitro digestion of DPYSL3 in cell lysate with purified calpain revealed a cleavage product identical to that observed in NMDA- and H2O2-treated cells, and its appearance was blocked by calpain inhibitors. Analysis of the DPYSL3 protein sequence revealed a possible cleavage site for calpain (Val-Arg-Ser) on the C-terminus of DPYSL3. Collectively, these studies demonstrate for the first time that DPYSL3 is a calpain substrate. The physiological relevance of the truncated DPYSL3 protein remains to be determined.
    Journal of Neurochemistry 11/2005; 95(2):466-74. · 3.97 Impact Factor
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    ABSTRACT: B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells.
    Toxicology and Applied Pharmacology 06/2005; 205(1):1-10. · 3.98 Impact Factor
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    ABSTRACT: Nickel compounds are carcinogenic and induce malignant transformation of cultured cells. Since nickel has low mutagenic potential, it may act predominantly through epigenetic mechanisms, including down-regulation of tumor suppressor genes. FHIT is a tumor suppressor gene whose expression is frequently reduced or lost in tumors and pre-malignant lesions. Previously, we have shown that the phosphohydrolase activity of Fhit protein, associated with its tumor suppressor action, is inhibited by nickel. In cells, such effect would assist in carcinogenesis. The latter could be further enhanced if nickel also lowered cellular levels of Fhit protein itself, e.g. by down-regulation of FHIT gene. To test this possibility, we determined Fhit protein and Fhit-mRNA levels in a nickel-transformed mouse cell line and in nickel-induced murine sarcomas. In B200 cells, derived by nickel treatment of BALB/c-3T3 cells and exhibiting a malignant phenotype, Fhit protein levels were 50% of those in the parental cells, while Fhit-mRNA expression remained unchanged. A decrease of up to > 90% in Fhit protein levels was also observed in 22 local sarcomas (mostly fibrosarcomas) induced by i.m. injection of nickel subsulfide in C57BL/6 and MT+ (C57BL/6 overexpressing metallothionein) mice, as compared with normal muscles. Moreover, Fhit was absent in 3 out of 10 sarcomas from MT+ mice and in 1 of 12 sarcomas from C57BL/6 mice. The lack of Fhit protein coincided with the absence of the Fhit-mRNA transcript in these tumors. However, in the other tumors, the decreased Fhit levels were not always accompanied by reduced expression of Fhit-mRNA. Thus, the observed lowering of Fhit protein levels is mostly associated with changes in mRNA expression and protein translation or turnover rates, and rarely with a full silencing of the gene itself. Overall, the decline of Fhit in cells or tissues malignantly transformed by nickel may indicate possible involvement of this effect in the mechanisms of nickel carcinogenesis.
    Molecular and Cellular Biochemistry 01/2004; 255(1-2):195-202. · 2.33 Impact Factor
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    ABSTRACT: FHIT (Fragile Histidine Triad) is a human tumor suppressor gene. The Fhit protein is believed to inhibit tumor growth by inducing apoptosis through interaction with diadenosine triphosphate (Ap(3)A). The latter is first sequestered and eventually hydrolyzed by Fhit to ADP and AMP. Thus, the balance between the cellular Ap(3)A level and Fhit enzymatic activity may affect cell death or survival. Increasing the Ap(3)A level, e.g., by inhibition of the enzyme, should prevent apoptosis and thus sustain tumorigenesis. To test if certain carcinogenic transition metals could inhibit the enzymatic activity of Fhit, purified human Fhit protein [30 nM in 1.25 mM poly(vinylpyrrolidone)], expressed in and isolated from E. coli, was incubated at pH 6.8 (50 mM HEPES buffer in 150 mM NaCl) with 120 microM Ap(3)A in the presence of 5 mM Mg(II) (activating cation) and 0-100 microM Ni(II), Cu(II), Zn(II), Cd(II), Co(II), Cr(III), As(III), or As(V). The reaction mixtures were analyzed by HPLC. The results revealed a strong inhibitory potential of Cu(II) [0.4], followed by Ni(II) [3.5] >or= Zn(II) [7.0] > Cr(III) [73] > Cd(II) [98] > Co(II) [432] [the numbers in brackets are IC(50) values, microM]. As(III) and As(V) had no effect. As revealed by spectrophotometry, mass spectrometry, and gel electrophoresis, the exceptionally strong inhibition by Cu(II) was associated with Fhit dimerization through formation of a disulfide bond. The other metals and also H(2)O(2) and NO did not cause the dimerization. Thus, the effect of Cu(II) must be due to its reaction with Cys-39 bearing the only thiol group in Fhit monomer. Since Cys-39 is not readily accessible in the Fhit molecule, the reaction is most likely facilitated by conformational changes which follow the coordination of Cu(II) by the surface histidines 35, 94, and/or 96. The observed inhibition of Fhit may be mechanistically involved in metal-mediated toxicity and carcinogenesis.
    Chemical Research in Toxicology 03/2002; 15(3):319-25. · 3.67 Impact Factor
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    ABSTRACT: The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase activity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap3A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.
    Molecular and Cellular Biochemistry 11/2001; 226(1-2):49-55. · 2.33 Impact Factor
  • T Pawelczyk, R Kowara, A Matecki
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    ABSTRACT: Protein kinase C-gamma (PKC-gamma) contains two cysteine-rich regions (Cys1, Cys2) responsible for interaction with phospholipids. However, previous experiments suggested that, only Cys1 represents the high affinity site involved in diacylglycerol-dependent activation of PKC-gamma. This raises the question whether Cys2 might participate in other functions of the PKC-gamma regulatory domain. The purpose of our studies was to examine the ability of Cys2 domain to bind cellular proteins. The Cys2 domain (residues 92-173) was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and purified. In order to investigate protein-protein interaction of Cys2 domain we used affinity column and an overlay assay. Our results demonstrate that the Cys2 domain of PKC-gamma binds several proteins from rat brain extracts. In the absence of phospholipids the Cys2 domain binds some proteins in the cytosolic fraction of rat brain, but no binding was detected with the proteins extracted from particulate fraction. Ca2+ at 1 microM concentration potentiated binding of cellular proteins to Cys2 domain. In the absence of Ca2+ the Cys2 domain binds proteins in the cytosolic fraction of rat brain in the presence of phosphatidylserine and to the lesser extend in the presence of phosphatidylinositol but neither phosphatidylcholine nor phosphatidylethanolamine. These results suggest that the Cys2 domain of PKC-gamma has the ability to interact with two classes of proteins. One class binds the Cys2 domain in the phosphatidylserine dependent fashion, and the other proteins bind Cys-2 domain in the Ca2+ dependent and phospholipid independent manner.
    Molecular and Cellular Biochemistry 07/2000; 209(1-2):69-77. · 2.33 Impact Factor
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    ABSTRACT: Protein kinase C- (PKC-) contains two cysteine-rich regions (Cys1, Cys2) responsible for interaction with phospholipids. However, previous experiments suggested that, only Cys1 represents the high affinity site involved in diacylglycerol-dependent activation of PKC-. This raises the question whether Cys2 might participate in other functions of the PKC- regulatory domain. The purpose of our studies was to examine the ability of Cys2 domain to bind cellular proteins. The Cys2 domain (residues 92-173) was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and purified. In order to investigate protein-protein interaction of Cys2 domain we used affinity column and an overlay assay. Our results demonstrate that the Cys2 domain of PKC- binds several proteins from rat brain extracts. In the absence of phospholipids the Cys2 domain binds some proteins in the cytosolic fraction of rat brain, but no binding was detected with the proteins extracted from particulate fraction. Ca2+ at 1 M concentration potentiated binding of cellular proteins to Cys2 domain. In the absence of Ca2+ the Cys2 domain binds proteins in the cytosolic fraction of rat brain in the presence of phosphatidylserine and to the lesser extend in the presence of phosphatidylinositol but neither phosphatidylcholine nor phosphatidylethanolamine.These results suggest that the Cys2 domain of PKC- has the ability to interact with two classes of proteins. One class binds the Cys2 domain in the phosphatidylserine dependent fashion, and the other proteins bind Cys-2 domain in the Ca2+ dependent and phospholipid independent manner.
    Molecular and Cellular Biochemistry 05/2000; 209(1):69-77. · 2.33 Impact Factor
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    ABSTRACT: The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit. Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells). The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity. The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A.
    Protein Expression and Purification 05/2000; 18(3):320-6. · 1.43 Impact Factor
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    ABSTRACT: Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-gamma was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UW spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UW spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5 bisphosphate (PIP2) or phosphatidyliinositol 4-phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. These data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at least overlapping. The site of PIP and PIP2 interaction with PKC-gamma is distinct from that of phorbol ester binding site.
    Acta biochimica Polonica 02/1999; 46(2):405-17. · 1.19 Impact Factor
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    ABSTRACT: in press