Regulation and control of gene expression and modulation of enzyme activity are critical aspects of cellular function. Although diverse mechanisms for regulating enzyme activities are well known, we report here a new paradigm for potential enzyme regulation in the trypanosomatids based on activators that are catalytically dead enzyme paralogs termed prozymes. Remarkably, these parasitic protozoa have independently evolved this mechanism in two different steps in the same essential biochemical pathway, the biosynthesis of spermidine and the subsequent hypusine modification of a critical lysine in the translation factor eIF5A. Gene duplication of both trypanosomatid AdoMetDC and DHS led to the evolution of an enzyme activation mechanism in which one paralog retained limited catalytic function and the other lost key catalytic residues, but retained the ability to oligomerize with the catalytic subunit to greatly enhance catalytic activity. A pivotal feature of this model is that observed activation by the prozyme component is dramatically large (1000–3000-fold) and is thus likely to result from cooperative structural changes.
The functional significance of prozyme activation of DHS has been clearly demonstrated by our studies. We show that both TbDHSc and TbDHSp are essential for the growth of mammalian blood form T. brucei and for infection of a mammalian host. Additionally, GC7, a known inhibitor of DHS, was found to have anti-trypanosomal activity. We demonstrate that the functional species of DHS in T. brucei is a heterotetrameric complex between TbDHSc and TbDHSp and that complex formation is required not only for full activity but also for stability of the proteins in the parasite. These data genetically and chemically validate T. brucei DHS as a potential drug target and demonstrate the importance of the functional heterotetrameric DHS complex.
Despite the similarities to the AdoMetDC example, significant differences in mechanism are also present. The two DHS subunits are not stable in T. brucei
unless in complex with each other. In contrast AdoMetDC prozyme and AdoMetDC levels are independent, and one can exist stably in excess over the other, which is a significant factor in AdoMetDC regulation in T. brucei
). T. brucei
up-regulates AdoMetDC prozyme protein levels in response to inhibition or knockdown of AdoMetDC. It remains to be determined if T. brucei
also regulates levels of DHSp to control deoxyhypusine formation in the parasite. Finally, although the AdoMetDC prozyme mechanism appears novel to the trypanosomatids, BLASTP analysis identified potential DHSp homologs in Entamoeba
species in addition to the trypanosomatids. Functional analysis is needed to determine whether the Entamoeba
DHSp paralog is also required to activate DHSc in this genus.
Our discovery that T. brucei
DHS is activated by a prozyme mechanism adds to the list of unusual and novel mechanisms that cells have evolved to regulate polyamine metabolism or modulate enzyme activity. Polyamine metabolism is tightly regulated in mammals, plants, and yeast, although interestingly, no regulation of DHS has been described (3
). Regulation occurs through common mechanisms such as transcriptional control but also through novel pathway-specific mechanisms. The intracellular turnover rate of ODC is controlled by expression of a protein inhibitor termed antizyme that targets ODC for degradation by the 26 S proteasome. Antizyme expression is in turn initiated by translational frame-shifting of antizyme mRNA when spermidine levels are high (32
), and it is further regulated by the antizyme inhibitor, which is itself an inactive paralog of ODC (33
). AdoMetDC expression is controlled by a small ribosome-stalling upstream open reading frame (uORFs) that is also sensitive to spermidine levels (34
). Trypanosomatids not only lack these mechanisms but unlike other eukaryotes are also unable to regulate RNA polymerase II transcription (35
). The protein coding genes typically lack introns (38
) and are transcribed as large polycistronic clusters, which undergo 5′-leader splicing of the pre-mRNA (39
). Regulation instead occurs during mRNA processing, mRNA degradation, translation, protein processing, and protein turnover (36
). Furthermore, as a consequence of the mRNA trans-splicing reaction, 5′-UTRs are short, and translational control by uORFs has not been observed. Thus, the driving force to evolve novel mechanisms to regulate the polyamine pathway in trypanosomatids may have been the paucity of other potential mechanisms. Given the large investment made by cells to control and regulate polyamine levels, together with the number of novel mechanisms that have been uncovered, it is clear that regulation of this pathway is a key cellular function.
Inactive paralogs have been identified in a wide variety of gene families in metazoan species, although they are most prevalent in the kinase, protease, sulfotransferase, and RAS-like protein families (40
). Inactive paralogs are perfectly poised to play regulatory roles, retaining the ability to bind both ligands and regulatory molecules. It has been shown that when duplicate genes evolve complementary mutations, the ability of cells to maintain both duplicates is enhanced, allowing novel function to evolve (44
), thus providing a platform for the evolution of a regulatory function. With the exception of pseudo-kinases, there is still limited functional data on the roles of inactive paralogs. Examples of regulation by both inhibitory and activating mechanisms have been described, although most of the examples involve inhibition or dominant negative effects. The sheer magnitude of the activation observed for T. brucei
DHS and AdoMetDC is unprecedented, and the observation that this occurs at two points in the same metabolic pathway is new.
In conclusion, the ability to regulate enzyme activity with a catalytically dead paralog provides cells with another tool for post-transcriptional regulation. Trypanosomatids represent the only known species where this regulatory strategy is available to potentially control the activity of DHS and AdoMetDC. Evolution of the prozyme mechanism in the trypanosomatids may have been driven by the need to control polyamine synthesis and function in an organism that lacks transcriptional control of gene expression and the frame-shifting and uORF-based mechanisms employed by many other eukaryotes. The discovery of this novel enzyme activation mechanism first for AdoMetDC and now for DHS powerfully confirms the importance of polyamines in the parasite, first exemplified by the discovery of trypanothione (6
). Our data suggest that the paradigm of enzyme activation by a catalytically dead paralog may be more widespread than currently known. Indeed, many additional examples of this mechanism for the regulation of enzyme function in eukaryotes are likely still undiscovered.