Product feedback regulation implicated in translational control of the Trypanosoma brucei S‐adenosylmethionine decarboxylase regulatory subunit prozyme

Abstract

Summary Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralog termed prozyme. Furthermore, prozyme protein levels were regulated in response reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3’UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3’UTR prozyme regulatory element sufficient to up regulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knockdown lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Polyamine and AdoMet metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

Genetic Validation of Trypanosoma brucei Glutathione Synthetase as an Essential Enzyme

“…However, quantitation of protein expression levels in the TbGS cDKO and RNAi lines suggests that Ͼ97% depletion of TbGS is required before trypanothione is depleted and cells begin to die, showing that a smallmolecule inhibitor of TbGS would need to nearly fully inhibit the enzyme. This is in contrast to ODC and AdoMetDC, where ϳ80% inhibition is sufficient (23,30).…”

“…Spermidine levels remain unchanged after depletion of TbGS, consistent with this result. In a previous study, we showed that decarboxylated AdoMet levels were inversely proportional to AdoMetDC prozyme levels, suggesting that elevated decarboxylated AdoMet levels lead to reduced prozyme expression (30). SpdSyn from human and Plasmodium has been shown to be feedback inhibited by methylthioadenosine, a reaction product of SpdSyn chemistry (51,52), and both spermidine and methylthioadenosine have been demonstrated by crystallographic studies to bind SpdSyn (53).…”

“…The polyamine and glutathione pathways are highly regulated in eukaryotic cells; however, the regulatory mechanisms that have been observed in other eukaryotes do not seem to be present in T. brucei. Instead, polyamine biosynthesis is uniquely regulated by the presence of a novel regulatory subunit of AdoMetDC, which activates the enzyme and also appears to be translationally regulated (23,30,31). Less is known about how the glutathione branch of the trypanothione biosynthetic pathway is regulated.…”

“…Regulatory control of the polyamine biosynthetic pathway in T. brucei appears to center on the AdoMetDC regulatory subunit prozyme, which responds to decarboxylated AdoMet depletion by upregulation of expression of prozyme and, to a lesser extent, ODC (23,30). Interestingly, knockdown of TbGS led to a decrease in prozyme and ODC protein levels and to an increase in ␥-GCS protein levels.…”

“…While T. brucei ␥-GCS has been characterized and shown to be essential for growth in T. brucei, gene knockdown did not lead to compensatory changes in the levels of the polyamine biosynthetic enzymes and no evidence for cross regulation between the polyamine and trypanothione arms of the pathway was found (22,28,29). Regulation has been shown to occur within the T. brucei polyamine biosynthetic pathway where gene knockdown or inhibition of S-adenosylmethionine decarboxylase (AdoMetDC), an essential enzyme in spermidine biosynthesis, led to increased protein expression of both the AdoMetDC regulatory subunit prozyme and ODC (23,24,30,31). The AdoMetDC regulatory protein prozyme is itself also an activator of AdoMetDC, as heterodimer formation with prozyme (an inactive paralog of AdoMetDC) increases enzymatic activity by 3 orders of magnitude.…”