Essentiality and Regulation of Deoxyhypusination in Trypanosoma brucei
Human African trypanosomiasis is caused by protozoan parasite Trypanosoma brucei. T. brucei and other trypanosomatids require spermidine for the formation of trypanothione, a unique thiol-redox factor. In other eukaryotes, spermidine is essential for the (deoxy)hypusination of eukaryotic initiation factor 5A (eIF5A). Hypusination, a post-translational modification, occurs via two enzymatic reactions. First, deoxyhypusine synthase (DHS) transfers the aminobutyl moiety of spermidine onto the eIF5A-lysine generating deoxyhypusine which is then hydroxylated by deoxyhypusine hydroxylase to yield the final modification, hypusine. Modified eIF5A has been shown to alleviate ribosome stalling on polyproline tracts. Human and yeast encode two isoforms of eIF5A but only one gene was identified in T. brucei (Tb927.11.740). Herein, I show that TbeIF5A and its modified lysine are essential for parasite growth by gene knockdown and complementation experiments. I have also identified potential proteins whose translation is regulated by eIF5A using proteomic profiling for proline-rich T. brucei proteins. Interestingly, unlike most eukaryotes, trypanosomatids encode two divergent paralogs of DHS (DHSp: Tb927.1.870 and DHSc: Tb927.10.2580), only one of which (DHSc) contains the key catalytic lysine. I showed that both DHS genes are essential for growth of bloodstream-form T. brucei using conditional gene knockouts, further establishing the requirement for deoxyhypusine in these parasites. My biochemical characterization of TbDHS showed that the two T. brucei paralogs form a heterotetrameric complex and that DHSp enhances the activity of recombinant DHSc by 3000-fold. While the essentiality of eIF5A and DHS is consistent with other eukaryotes, the finding that the functional DHS complex is composed of an impaired catalytic subunit (DHSc) and a catalytically dead paralog (termed a prozyme) is novel. This mechanism reiterates the activation and regulation of S-adenosylmethionine decarboxylase by a catalytically dead paralog (AdoMetDC prozyme) in the trypanosomatids, and remarkably, it has independently evolved for two enzymes within the trypanosomatid spermidine biosynthetic pathway. T. brucei seemingly lack several classical eukaryotic transcriptional regulation mechanisms which creates selective pressure to evolve novel strategies to regulate enzyme function. We postulate that many additional examples of 'prozymes' remain to be discovered in the trypanosomatid parasites.
Lysine, Peptide Initiation Factors, Protein Processing, Post-Translational, Protozoan Proteins, RNA-Binding Proteins, Trypanosoma brucei brucei
University of Texas Southwestern Medical Center
Networked Digital Library of Theses and Dissertations