AbstractTrypanosomatids are an unusual early-branching group of eukaryotic organisms in which many biological pathways differ extensively from the same pathways seen in other eukaryotes. Trypanosomatids include several medically relevant species such as Trypanosoma brucei, which is the causal agent of the tropical disease African sleeping sickness, and pathogenic Leishmania species causing different manifestations of leishmaniasis. New medicines are very much needed to treat these neglected tropical diseases. In eukaryotes, cytochromes c and c1 are essential components of a typical mitochondrial electron transport chain, which is used for energy production. Generally, the activity of c-type cytochromes is determined by the covalent attachment of heme to two cysteine residues in a heme-binding motif. Mitochondrial cytochromes found in trypanosomatids are unique because they contain a single cysteine in the heme-binding motif. For the heme to become attached to cysteine(s) in a heme-binding motif, c-type cytochromes must undergo post-translation modification (or maturation). Previous genome analyses, in contrast to other eukaryotes, provided no evidence for the existence of the protein(s) responsible for catalysing this post-translational modification in trypanosomatids. A study that identified this maturation system would solve this puzzle and could provide an opportunity to identify a novel therapeutic target for a group of medically important parasitic protists.
In the mitochondrial intermembrane space of eukaryotes, other than trypanosomatids, either System I or III is used to attach heme to c-type cytochromes. One of my early analyses was to determine the distribution and key functional residues of both System I and III. The aim was to understand the types of motifs that might be essential for heme attachment in trypanosomatids’ cytochrome c maturation system. The search for a candidate maturation protein was carried out using proteomic sift between trypanosomatids and Phytomonas proteomes. Candidate proteins conserved in trypanosomatids but not in Phytomonas were compiled and analysed to assess whether they contain conserved key motifs of either System I or III. In probing the evolutionary biochemistry of cytochrome c maturation, an unexpected variation in the distribution of Systems I versus III was noticed in different eukaryotic clades. Another significant outcome from the analysis was the identification of a candidate cytochrome c maturation in the trypanosomatids. The overall architecture of this candidate suggested a divergent System III. A combination of laboratory techniques was used to experimentally prove that this candidate protein functions as a catalyst for the maturation of the trypanosomatids c-type cytochrome. These techniques include protein expression and purification followed by heme staining and UV-Vis spectroscopy to detect heme attachment.
In the second part of my research, I focused on other aspects of the unusual metabolic biochemistry in other protists. In particular, I used bioinformatics and literature-led approaches to analyse mitochondrial metabolism in Naegleria species. N. gruberi, distantly related to the Euglenozoa is one of the few eukaryotes with extreme versatility to its energy metabolism and capable of apparent switching between oxidative phosphorylation, anaerobic respiration and fermentation linked to H2 generation. The focus of the bioinformatics investigation was to examine the capability of N. gruberi to encode for two well-known anaerobic enzymes; acetyl-CoA synthetase and nitrite reductase. Findings have shown that N. gruberi candidate acetyl-CoA synthetase does not possess the functional key residues used to characterise the acetyl-CoA synthetase family. Thus, it is likely that N. gruberi candidate protein is involved in an alternative catalytic pathway. On the other hand, N. gruberi candidate nitrite reductase has all the essential residues and overall structure previously identified to be critical for nitrite reductase function. This indicates that it is likely to contribute to anaerobic respiration under appropriate environmental conditions. Another area of focus was to examine the cryptic peroxisomal targeting of the metabolic enzymes. The outcome of this analysis shows N. gruberi cryptic PTS1 motifs, in several metabolic enzymes including some of the main glycolytic enzymes, can confer peroxisomal targeting, which suggests that the function of peroxisomes can be more versatile than previously expected.
|Date of Award||2023|
|Supervisor||Michael Ginger (Co-Supervisor) & Martin Carr (Co-Supervisor)|