Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

Abstract

The fate of the [methyl-14C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-14C]methionine, [U-14C]methionine, S-[methyl-14C]AdoMet or [33S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-14C] and [U-14C]methionine to [35S]methionine. After 4-h incubation with [U-14C]methionine, [methyl-14C]methionine or [35S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-14C] or [methyl-14C]methionine in the presence of cycloheximide (50 micrograms/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [35S]methionine and cycloheximide. [Methyl-14C] and [U-14C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-14C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-14C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-14C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.

In vivo efficacies of 5'-methylthioadenosine analogs as trypanocides

“…In trypanosomes its role is enhanced by the absolute requirement for preformed purines and the extensive use of methionine for AdoMet synthesis as opposed to incorporation into protein (12). AdoMet in trypanosomes is used most extensively in protein and lipid methylation as well as in polyamine synthesis (12,13). HETA is a substrate analog of MTA cleavable into adenine and 5-(hydroxyethylthio) ribose-1-phosphate (HETR), the hydroxyethyl analog of methylthioribose-1-phosphate (4).…”

Polyamine Metabolism as Chemotherapeutic Target in Protozoan Parasites

“…Studies with DFMO-treated trypanosomes also demonstrated that AdoMet synthetase, carefully regulated in mammalian cells, continued to function after putrescine production (and the need for AdoMet) had ceased [9]. This resulted in significant accumulation of AdoMet and dAdoMet, and was accompanied by increases in protein methylation [51].…”

Extremely rapid turnover of S‐adenosylmethionine decarboxylase in Crithidia fasciculata

“…Decarboxylated AdoMet provides the aminopropyl moiety for the synthesis of spermidine from putrescine (and of spermine from spermidine in organisms containing also this polyamine). In addition to providing the aminopropyl group for the synthesis of spermidine, AdoMet in the trypanosome parasites is utilized as the methyl donor for transmethylation reactions as well as for salvage of adenine and adenosine [7,8]. The decarboxylation of AdoMet has a pivotal role in the switching between cellular methylation and polyamine biosynthesis in the parasites [9].…”