Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1.

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.     

DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: the lack of this suppression in p53MUT cells contributes to apoptosis

DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53(WT) colorectal carcinoma cells a long term cell cycle arrest and in p53MUT cells a transient arrest followed by apoptosis (Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherübl, H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Int. J. Cancer 101, 23-31; Bhonde, M. R., Hanski, M. L., Notter, M., Gillissen, B. F., Daniel, P. T., Zeitz, M., and Hanski, C. (2006) Oncogene 25, 165-175). The mechanism of the p53-independent apoptosis still remains largely unclear. Here we used five p53WT and five p53MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53MUT cells than in the cell cycle-arrested p53WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53MUT cells after SN-38 treatment contributes to DNA damage-induced apoptosis, whereas their suppression in p53WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53MUT cells activates the checkpoint and contributes to apoptosis.     

Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in Epstein-Barr Virus-Infected Cells

All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.     

Multiple sclerosis - remyelination failure as a cause of disease progression

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) that affects worldwide about 2.5 million people. The morphological correlates of the disease are multiple lesions in brain and spinal cord which are characterized by demyelination, inflammation, gliosis and axonal damage. The underlying cause for the permanent neurological deficits in MS patients is axonal loss. Demyelinated axons are prone to damage due to the lack of trophic support by myelin sheaths and oligodendrocytes, as well as the increased vulnerability to immune mediated attacks. Remyelination occurs, but especially in chronic lesions is frequently limited to a small rim at the lesion border. Current treatment strategies are based on anti-inflammatory or immunomodulatory drugs and have the potential to reduce the numbers of newly evolving lesions, although as yet no treatment strategy exists to influence or prevent the progressive disease phase. Therefore, the development of neuroprotective treatment options, such as the promotion of endogenous remyelination is an attractive strategy. A prerequisite for the development of such new treatments is the understanding of the mechanisms leading to remyelination and the reasons for insufficient endogenous repair in chronic MS. This review will therefore provide an overview of the current concepts regarding remyelination in the rodent and human CNS. We will also summarize a selected number of inhibitory pathways and non-disease related factors which may contribute to remyelination failure in chronic MS.     

Biologic characterization of human bone tumors

The etiology of aneurysmal bone cyst is still unknown. Most theories of the histogenesis of this lesion assume a vascular origin and speculation has focused on the characteristic pseudoendothelial lining of the cyst walls. In the present study, this structure has been subjected to enzyme histochemical, electron microscopical, and immunohistochemical investigation. Of the enzymes tested only alkaline phosphatase was present in the cyst lining. Electron microscopy revealed fibroblast-like cells covering the walls of cystic cavities, but no genuine endothelium, basement membranes or pericytes were identified. For the immunohistochemical studies a panel of poly- and monoclonal antibodies against HLA-DR antigens, mature and immature macrophages/histiocytes, smooth muscle fibers and endothelial cells, as well as the lectin Ulex europaeus I agglutinin were used. None of these markers demonstrated the presupposed vascular characteristics in the cells constituting the pseudoendothelial lining of the cyst walls. Despite current theories to the contrary, it was concluded that aneurysmal bone cyst is unlikely to originate from the vascular system, and that a new concept of its pathogenesis must be sought.     

A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy.

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.     

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.     

The Epstein-Barr virus BRRF1 protein, Na, induces lytic infection in a TRAF2- and p53-dependent manner

The Epstein-Barr virus (EBV) BRRF1 lytic gene product (Na) is encoded within the same immediate-early region as the BZLF1 (Z) and BRLF1(R) gene products, but its role during EBV infection has not been well defined. We previously showed that Na cooperates with the R protein to induce lytic gene expression in latently infected EBV-positive 293 cells, and in some EBV-negative cell lines it can activate the Z promoter in reporter gene assays. Here we show that overexpression of Na alone is sufficient to induce lytic gene expression in several different latently infected epithelial cell lines (Hone-Akata, CNE2-Akata, and AGS-Akata), while knockdown of endogenous Na expression reduces lytic gene expression. Consistent with its ability to interact with tumor necrosis factor receptor-associated factor 2 (TRAF2) in a yeast two-hybrid assay, we demonstrate that Na interacts with TRAF2 in cells. Furthermore, we show that TRAF2 is required for Na induction of lytic gene expression, that Na induces Jun N-terminal protein kinase (JNK) activation in a TRAF2-dependent manner, and that a JNK inhibitor abolishes the ability of Na to disrupt viral latency. Additionally, we show that Na and the tumor suppressor protein p53 cooperate to induce lytic gene expression in epithelial cells (including the C666-1 nasopharyngeal carcinoma cell line), although Na does not appear to affect p53 function. Together these data suggest that Na plays an important role in regulating the switch between latent and lytic infection in epithelial cells and that this effect requires both the TRAF2 and p53 cellular proteins.