Analysis of protein kinases regulating the Trypanosoma brucei cell cycle

Q Science (General);QR Microbiology

Thomas, Elizabeth Baby ; Hammarton, Tansy

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Trypanosoma brucei, trypanosomes, cell cycle, kinase, polo-like kinase, PLK, screen, RNAi;   

Others  :  http://theses.gla.ac.uk/6229/1/2015ThomasPhD.pdf

来源: University of Glasgow

【 摘 要 】

Trypanosoma brucei spp. are protozoan parasites that cause Human African Trypanosomiasis in humans, and Nagana in cattle. These diseases are mostly fatal if left untreated and there is an urgent need for safe, effective drugs that can be easily administered. T. brucei has a complex cell cycle, the regulation of which appears to be divergent compared to other model eukaryotes. This implies that the regulators of the T. brucei cell cycle could be exploited as a source of novel drug targets. One such cell cycle regulator of interest is T. brucei polo-like kinase (TbPLK), a serine/threonine protein kinase thought to diverge from its canonical functions in eukaryotic mitosis and be mainly involved in the duplication of the parasite’s cytoskeletal structures. This study sought to investigate how the activity and localisation of TbPLK is regulated in procyclic form (PCF) and bloodstream form (BSF) parasites. A second aim of this study was to identify novel protein kinases (PKs) which regulate the T. brucei cell cycle by screening part of a kinome-wide RNA interference (RNAi) library of BSF cell lines, that has recently been established (Jones et al. 2014). The cell lines had already been assessed for proliferation defects upon RNAi induction by using an Alamar blue viability assay. In this study, the cell lines which displayed proliferation defects were further screened for cell cycle defects using growth curves and DAPI staining, to identify as yet uncharacterised protein kinases required for T. brucei cell cycle regulation. 50 PKs had been shown to be required for viability in vitro and were screened for potential cell cycle roles. Of these, 25 were identified as potential cell cycle regulators, 15 of which were detected for the first time. The majority of the hits were deemed to be involved in either just cytokinesis, or cytokinesis in combination with kinetoplast duplication or mitosis, with surprisingly few in G1/S. Knockdown of a number of these putative cell cycle PKs induced cell death signifying their potential as drug targets. Indeed, one of the hits, CLK1, was genetically validated as a potential drug target in a mouse model. The identification of these putative cell cycle kinases has also provided valuable starting points by which the signalling pathways that regulate the cell division cycle of these parasitic organisms can be elucidated.