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Expression pattern, regulation, and functions of methionine adenosyltransferase 2beta splicing variants in hepatoma cells.

ABSTRACT: Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5'-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2.Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays.MAT2beta is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-alpha, which induces MAT2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-alpha induced the promoter activity of MAT2beta V1, likely via nuclear factor kappaB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-alpha-induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types.Different variants of MAT2beta regulate growth and death, which broadens their importance in biology.

SUBMITTER: Yang H

PROVIDER: S-EPMC2409110 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Expression pattern, regulation, and functions of methionine adenosyltransferase 2beta splicing variants in hepatoma cells.

Yang Heping H Ara Ainhoa Iglesias AI Magilnick Nathaniel N Xia Meng M Ramani Komal K Chen Hui H Lee Taunia D TD Mato José M JM Lu Shelly C SC

Gastroenterology 20071018 1

<h4>Background & aims</h4>Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5'-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: ...[more]

PMID: 18045590

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Project description:BackgroundTuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.ResultsThe gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM.ConclusionThe methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.

Dataset Information

Expression pattern, regulation, and functions of methionine adenosyltransferase 2beta splicing variants in hepatoma cells.

Publications

Expression pattern, regulation, and functions of methionine adenosyltransferase 2beta splicing variants in hepatoma cells.

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