Investigating the role of rhomboid proteases in the development of Dictyostelium discoideum
Dictyostelium discoideum is a model eukaryotic organism with many equivalent cellular mechanisms to humans. In this project Dictyostelium has been used to help investigate the role of rhomboid proteins. Bioinformatics tools were used to identify four potential rhomboids encoded within the Dictyostelium genome that contain the residues necessary for proteolytic activity. To identify their functions, three null mutants rhmA- , rhmB- and rhmC- have been constructed, and their growth and development observed. It was found that development was unaltered following deletion of rhmC, whereas attempts to knock out the putative mitochondrial rhmD were unsuccessful. rhmA and rhmB null mutants gave rise to pleiotropic effects, rhmA altering the response to chemoattractants and demonstrating decreased motility in general. It also showed altered mitochondrial morphology which is consistent with phototaxis defects in slugs. rhmB null cells had slower growth rates, reaching stationary phase at a reduced density; a delayed response to folic acid stimulation; larger cell size in the unicellular phase and altered stalks in the multicellular phase. These results correspond with qPCR analysis, in which RhmA and RhmB transcript levels are highest during the multicellular development phase. Dictyostelium is also a useful non-animal eukaryote for testing novel compounds and dissecting cell regulatory molecular networks. In this thesis I have used this social amoeba to test the effect of a series of arylboronic acids and a novel compound, cardiosulfa, on development, chemotaxis and viability of Dictyostelium. The arylboronic acids were investigated previously by collaborators for serine protease inhibition and urokinase-type plasminogen activator (uPA) inhibition, both in vitro and in vivo. In those biochemical assays, three compounds, BC11, SR3 and BC57 displayed micromolar inhibition of uPA with an excellent selectivity profile over related proteases (Smith et al., 2012). Previous studies suggested that cardiosulfa, disrupted heart development in the zebrafish. Gene expression profiling studies showed that cardiosulfa induces high transcription levels of members of the aryl hydrocarbon receptor (AhR) family (Ko et al. 2009). These test compounds reported to show effects in vivo also disturbed cell adhesion and migration, without having an effect on Dictyostelium viability. Networks of predicted activity were constructed to establish molecular mechanisms for cardiosulfa and arylboronic acids analogues. In conclusion this project provides the first insight into the molecular and cellular functions of rhomboid proteins, potential aryl hydrocarbon receptor and urokinase-type plasminogen activator by using Dictyostelium as an early biomedical model to study these effects in eukaryotes.
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