Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry and Molecular Biology

Advisor

Morris, James C

Committee Member

Paul , Kimberly S

Committee Member

Sehorn , Michael G

Committee Member

Smith , Kerry S

Abstract

Trypanosoma brucei, a unicellular eukaryotic parasite, is the causative agent of African sleeping sickness in sub-Saharan Africa. The parasite encounters two main environments as it progresses through its life cycle: the tsetse fly and the mammalian
bloodstream. Nutrient availability is distinct in the two environments, requiring the parasite to utilize diverse metabolic pathways to efficiently produce ATP for survival. Bloodstream form parasites (BSF), residing in a glucose rich environment, rely solely on
glycolysis for energy, while procyclic form (PF) parasites metabolize readily available proline and threonine in addition to glucose.
T. brucei expresses two hexokinases, the first enzyme in the glycolytic pathway, that are 98.5% identical at the nucleotide level and 98% similar at the amino acid level. These two enzymes, TbHK1 and TbHK2, are differentially expressed in both BSF and PF
parasites. Here, I identify a means of regulation of TbHK1 gene expression and a novel mechanism for regulating TbHK1 enzyme activity. Lastly, I have characterized, the bioflavonoid quercetin, a small molecule regulator TbHK1.
Mechanisms involved in regulating TbHK1 and TbHK2 gene expression have not been extensively studied. I have found that TbHK1 uses differential polyadenylation to
regulate gene expression in variable environmental conditions. A recent study by Siegel et al. (2010), revealed that TbHK1 contains seven predominant polyadenylation sites, whereas, TbHK2 contains only one. Using a reporter gene system to assess transcript level and protein expression, I have determined that these seven different 3'UTR lengths result in differential steady state transcript abundance and expression level, dependent on nutrient availability.
Further, I have identified a novel means of regulation of TbHK1 enzyme activity. Enzymatic studies of recombinant TbHK1 reveal that TbHK is inactivated under acidic conditions. However, the addition of glycerol-3-phosphate to the reaction at acidic pH maintained T. brucei hexokinase activity. I propose this regulation may play an important role in the biology of the parasite during differentiation and subsequent acidification of glycosomes, the peroxisomal like organelle that houses glycolytic enzymes.
Lastly, the TbHKs were identified as possible drug targets because they are essential to the BSF parasite and only ~30% similar to human glucokinase, the human equivalent of TbHK1. We have found that quercetin, a known trypanocide, is a potent inhibitor of TbHK1. Further, taking advantage of the fluorescent nature of the compound, I found that the compound localizes to the same subcellular compartment that houses TbHK1 and interacts with the protein near the active site as a mixed inhibitor.

Included in

Biochemistry Commons

Share

COinS