Optimizing the Inhibition of a Uniquely Composed Trypanosoma brucei F1-ATPase

0488324 - BC 2018 RIV CZ eng O - Others
Gahura, Ondřej - Váchová, H. - Panicucci, Brian - Walker, J.E. - Zíková, Alena
Optimizing the Inhibition of a Uniquely Composed Trypanosoma brucei F1-ATPase.
2014
R&D Projects: GA MŠMT LL1205
Institutional support: RVO:60077344
Keywords : inhibition * ATPase * protein
OECD category: Biochemistry and molecular biology
http://www.parazitologie.cz/protozoologie/Protodny2014/JPD_sbornik_2014.pdf

The transition of the parasitic Trypanosoma brucei between its invertebrate and vertebrate hosts is associated with substantial bioenergetic pathway changes. While substrate and oxidative phosphorylation (OXPHOS) provide the main source of ATP in the procyclic stage (PS), increased glycolysis of abundant glucose in the bloodstream form (BS) compensates for the absence of OXPHOS - requiring the F1Fo-ATPase to maintain the mitochondrial membrane potential at the expense of ATP. A widespread natural protein inhibitor of F1Fo-ATPase activity (TbIF1) is expressed in PS, while its ectopic expression is lethal in BS. To characterize TbIF1 inhibition, we isolated the F1-ATPase from PS by two-step chromatography. Besides the conserved eukaryotic components (3 3), the complex contains an additional trypanosomatid-specific protein, p18. The previously reported subunit cleavage was confirmed and modeled to a region presumed to form a loop between the crown and NTP-binding domains, a unique feature of F1-ATPase in Kinetoplastids. Furthermore, several recombinant TbIF1 mutants were characterized to determine their dissociation constants and oligomerization properties. While the C-terminal deletion of TbIF1 prevents homodimerization, it does not disrupt the pH sensitivity as it does in bovine IF1. Importantly, TbIF1 cannot inhibit bovine F1-ATPase and vice versa, strengthening the differences between the parasite and mammals. The established purification of a uniquely composed F1-ATPase is suitable for structure resolution by X-ray crystallography. Given the non-conventional function of F1-ATPase in BS and the F1-TbIF1 binding data, we propose that the structure could be exploited to design specific inhibitors for potential use in therapeutics.

Permanent Link: http://hdl.handle.net/11104/0282936