The infectious lifecycle stage of the African trypanosome, Trypanosoma brucei, relies exclusively on glycolysis for ATP generation. The parasite responds to dynamic environmental cues to regulate this pathway. The goal of this application is to resolve the mechanisms that regulate localization of the essential glycolytic enzymes, T. brucei hexokinase 1 and 2, with a particular emphasis on understanding the glucose-sensitive subcellular localization of the proteins. We have previously demonstrated that T. brucei hexokinase 2, which was heretofore believed to be limited in its distribution to a peroxisome-like organelle called the glycosome, has in addition a life-cycle dependent extra-glycosomal localization. In the bloodstream form parasites, the protein was found in the flagellum while in the insect stage the protein was found proximal to the basal bodies. Our preliminary data suggests that the localization is dependent on environmental glucose availability and that the hexokinases are associated with a detergent-insoluble fraction of the mitochondria. Using both ectopic expression of tagged proteins and immunochemistry in collaboration with microscopy and biochemical approaches, we will identify the enzyme domains that are required for localization. Additionally, we will begin to resolve the mechanisms required for glucose-dependent localization, with a particular focus on the association of the hexokinases with the parasite mitochondrion. Through these studies, new ways of targeting glucose metabolism, an essential parasite pathway, will be identified.
The research proposed here is important to public health because understanding the biology of the essential trypanosome hexokinases is the first step in the identification of new targets for desperately needed therapeutic development. Additionally, this work will expand our understanding of the fundamental cellular processes of nutrient and environmental sensing, topics that are supported by the mission of the NIH.
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