Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry and Molecular Biology

Advisor

Morris, James C

Committee Member

Marcotte, Jr. , William R

Committee Member

Temesvari , Lesly A

Committee Member

Feltus , Frank A

Abstract

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and nagana in livestock. The parasite inhabits multiple environmental niches including the bloodstream of the mammalian host and the mid-gut of the tsetse fly vector. While in the bloodstream of its mammalian host, the organism depends solely on glycolysis for production of ATP. My studies focus on the first enzyme in glycolysis, hexokinase.
T. brucei has two hexokinases, TbHK1 and TbHK2 that are 98.5% identical at the nucleotide level. The hexokinases are expressed in the glycosomes of both procyclic form and bloodstream form parasites. Glycosomes are peroxisome-like organelles that house glycolysis, along with other functions, in trypanosomes. Here I present evidence that PEX7, a known glycosome import protein in other systems, may be responsible for the import of TbHKs into the glycosome. Further, I report that, in addition to the glycosome, TbHK2 also localizes to the flagellum of BSF parasites and to two punctate bodies proximal to the basal body in PF parasites.
TbHK1 has also been validated as a drug target for treatment of African trypanosomiasis. A high throughput screen has been conducted in which 220,233 compounds were tested against TbHK1 for inhibitory effects. In this screen, ebselen was identified as a potent inhibitor of TbHK1. Ebselen is a known cysteine-reactive seleno-organic compound. Here I show that while ebselen does indeed bind the Cys residues of TbHK1, this binding does not result in inhibition of the enzyme. Lastly, I show that three Cys residues are necessary for the activity of TbHK1. These studies have improved our knowledge of the localization, inhibition, and oligomerization of TbHKs.

Included in

Biochemistry Commons

Share

COinS