The effect of trichloroethylene metabolites on the hepatic vitamin B12-dependent methionine salvage pathway and its relevance to increased excretion of formic acid in the rat

The industrial solvent trichloroethylene (TCE) and its two major metabolites trichloroethanol (TCE-OH) and trichloroacetic acid (TCA) cause formic aciduria in male F344 rats. Prior treatment of male F344 rats with 1-aminobenzotriazole a cytochrome P450 inhibitor, followed by TCE (16mk/kg, po), completely prevented formic aciduria, but had no effect on formic acid excretion produced by TCA (8 or 16 mg/kg, po), suggesting TCA may be the proximate metabolite producing this response. Dow and Green reported an increase in the concentration of 5-methyltetrahydrofolate (5-MTHF) in the plasma of rats treated with TCE-OH, suggesting a block in the cycling of 5-MTHF to tetrahydrofolate (THF). This pathway is under the control of the vitamin B12-dependent methionine salvage pathway. We therefore treated rats with three daily doses of methylcobalamin (CH3Cbl) or hydroxocobalamin (OHCbl), a cofactor for methionine synthase, or L-methionine, followed by TCE (16 mg/kg) to determine if they could alleviate the formic aciduria. These pretreatments only partially reduced the excretion of formic acid in the urine. Although prior treatment with S-adenosyl-L-methionine had no effect on formic acid excretion. Consistent with these findings, the activity of methionine synthase in the liver of TCE-treated rats was not inhibited. Transcriptomic analysis of the liver-identified nine differential expressed genes, of note, was downregulation of Lmbrd1 involved in the conversion of vitamin B12 into CH3Cbl, a cofactor for methionine synthase. Our findings indicate that the formic aciduria produced by TCE-OH and TCA may be the result of a block in the recycling of 5-MTHF to THF, the effect on the methionine salvage pathway being a secondary response following acute exposure.

Pharmacological polyamine catabolism upregulation with methionine salvage pathway inhibition as an effective prostate cancer therapy

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.

Two Distinct Aerobic Methionine Salvage Pathways Generate Volatile Methanethiol inRhodopseudomonas palustris

ABSTRACT5′-Methyl-thioadenosine (MTA) is a dead-end, sulfur-containing metabolite and cellular inhibitor that arises fromS-adenosyl-l-methionine-dependent reactions. Recent studies have indicated that there are diverse bacterialmethioninesalvagepathways (MSPs) for MTA detoxification and sulfur salvage. Here, via a combination of gene deletions and directed metabolite detection studies, we report that under aerobic conditions the facultatively anaerobic bacteriumRhodopseudomonas palustrisemploys both an MTA-isoprenoid shunt identical to that previously described inRhodospirillum rubrumand a second novel MSP, both of which generate a methanethiol intermediate. The additionalR. palustrisaerobic MSP, a dihydroxyacetone phosphate (DHAP)-methanethiol shunt, initially converts MTA to 2-(methylthio)ethanol and DHAP. This is identical to the initial steps of the recently reported anaerobic ethylene-forming MSP, the DHAP-ethylene shunt. The aerobic DHAP-methanethiol shunt then further metabolizes 2-(methylthio)ethanol to methanethiol, which can be directly utilized by O-acetyl-l-homoserine sulfhydrylase to regenerate methionine. This is in contrast to the anaerobic DHAP-ethylene shunt, which metabolizes 2-(methylthio)ethanol to ethylene and an unknown organo-sulfur intermediate, revealing functional diversity in MSPs utilizing a 2-(methylthio)ethanol intermediate. When MTA was fed to aerobically growing cells, the rate of volatile methanethiol release was constant irrespective of the presence of sulfate, suggesting a general housekeeping function for these MSPs up through the methanethiol production step. Methanethiol and dimethyl sulfide (DMS), two of the most important compounds of the global sulfur cycle, appear to arise not only from marine ecosystems but from terrestrial ones as well. These results reveal a possible route by which methanethiol might be biologically produced in soil and freshwater environments.IMPORTANCEBiologically available sulfur is often limiting in the environment. Therefore, many organisms have developed methionine salvage pathways (MSPs) to recycle sulfur-containing by-products back into the amino acid methionine. The metabolically versatile bacteriumRhodopseudomonas palustrisis unusual in that it possesses two RuBisCOs and two RuBisCO-like proteins. While RuBisCO primarily serves as the carbon fixation enzyme of the Calvin cycle, RuBisCOs and certain RuBisCO-like proteins have also been shown to function in methionine salvage. This work establishes that only one of theR. palustrisRuBisCO-like proteins functions as part of an MSP. Moreover, in the presence of oxygen, to salvage sulfur,R. palustrisemploys two pathways, both of which result in production of volatile methanethiol, a key compound of the global sulfur cycle. When total available sulfur was plentiful, methanethiol was readily released into the environment. However, when sulfur became limiting, methanethiol release decreased, presumably due to methanethiol utilization to regenerate needed methionine.

Metabolic Regulation as a Consequence of Anaerobic 5-Methylthioadenosine Recycling inRhodospirillum rubrum

ABSTRACTRhodospirillum rubrum possesses a novel oxygen-independent, aerobic methionine salvage pathway (MSP) for recycling methionine from 5-methylthioadenosine (MTA), the MTA-isoprenoid shunt. This organism can also metabolize MTA as a sulfur source under anaerobic conditions, suggesting that the MTA-isoprenoid shunt may also function anaerobically as well. In this study, deep proteomics profiling, directed metabolite analysis, and reverse transcriptase quantitative PCR (RT-qPCR) revealed metabolic changes in response to anaerobic growth on MTA versus sulfate as sole sulfur source. The abundance of protein levels associated with methionine transport, cell motility, and chemotaxis increased in the presence of MTA over that in the presence of sulfate. Purine salvage from MTA resulted primarily in hypoxanthine accumulation and a decrease in protein levels involved in GMP-to-AMP conversion to balance purine pools. Acyl coenzyme A (acyl-CoA) metabolic protein levels for lipid metabolism were lower in abundance, whereas poly-β-hydroxybutyrate synthesis and storage were increased nearly 10-fold. The knownR. rubrumaerobic MSP was also shown to be upregulated, to function anaerobically, and to recycle MTA. This suggested that other organisms with gene homologues for the MTA-isoprenoid shunt may also possess a functioning anaerobic MSP. In support of our previous findings that ribulose-1,5-carboxylase/oxygenase (RubisCO) is required for an apparently purely anaerobic MSP, RubisCO transcript and protein levels both increased in abundance by over 10-fold in cells grown anaerobically on MTA over those in cells grown on sulfate, resulting in increased intracellular RubisCO activity. These results reveal for the first time global metabolic responses as a consequence of anaerobic MTA metabolism compared to using sulfate as the sulfur source.IMPORTANCEIn nearly all organisms, sulfur-containing byproducts result from many metabolic reactions. Unless these compounds are further metabolized, valuable organic sulfur is lost and can become limiting. To regenerate the sulfur-containing amino acid methionine, organisms typically employ one of several variations of a “universal” methionine salvage pathway (MSP). A common aspect of the universal MSP is a final oxygenation step. This work establishes that the metabolically versatile bacteriumRhodospirillum rubrumemploys a novel MSP that does not require oxygen under either aerobic or anaerobic conditions. There is also a separate, dedicated anaerobic MTA metabolic route inR. rubrum. This work reveals global changes in cellular metabolism in response to anaerobic MTA metabolism compared to using sulfate as a sulfur source. We found that cell mobility and transport were enhanced, along with lipid, nucleotide, and carbohydrate metabolism, when cells were grown in the presence of MTA.