Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei . In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S -adenosylmethionine decarboxylase ( Tb AdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T . brucei an alternative regulatory strategy based on Tb AdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of Tb ... More
Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei . In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S -adenosylmethionine decarboxylase ( Tb AdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T . brucei an alternative regulatory strategy based on Tb AdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of Tb AdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which Tb AdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In Tb AdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either Tb AdoMetDC or an active site mutant, suggesting that Tb AdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of Tb AdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T . brucei . These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology.Author summaryTrypanosoma brucei is a single-celled eukaryotic pathogen and the causative agent of human African trypanosomiasis (HAT). Polyamines are organic polycations that are essential for growth in T . brucei to facilitate protein translation and to maintain redox homeostasis. The pathway is the target of eflornithine, a current frontline therapy for treatment of HAT. Polyamine biosynthetic enzymes are regulated at multiple levels in mammals (e.g. transcription, translation and protein turnover), but in contrast, T . brucei lacks these mechanisms. Instead in T . brucei a central enzyme in polyamine metabolism called AdoMetDC must form a complex with a sister protein (termed a pseudoenzyme) to be active. Herein, we show that cellular levels of this sister protein we call prozyme are in turn feedback regulated by both AdoMetDC and by its reaction product in response to cell treatments that reduce pathway output. This regulatory paradigm highlights how pseudoenzymes can evolve to play an important role in metabolic pathway regulation and in organismal fitness.
