Effects of carboxylmethylation and polyamine synthesis inhibitors on methylation of Trypanosoma brucei cellular proteins and lipids

The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-a-difluoromethylornithine (Omidyl, DFMO) the AdoMet decarboxylase inhibitor 5'-[[(Z)-4-amino-2-butenyl]-methylamino)-5'-deoxyadenosin or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [S-35] or [U-C-14]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fractions. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 mu M) and sinefugin (2 nM) on [S-35] and [U-C-14]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U-C-14]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-C-14]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-C-14]methionine as AdoMet. Exogenous AdoMet had no effect on [S-35]methionine incorporation. The agents studied are curative for African trypanosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.