Investigating Invadolysin's activity: discerning mechanism and cellular roles

Abstract

Invadolysin is a novel metalloprotease, which is conserved amongst metazoans and was first identified in the Heck laboratory. Proteases play a variety of roles in normal physiology. Invadolysin is essential for life in Drosophila. Invadolysin has been shown to be essential for cell division and cell migration. Invadolysin is the only metalloprotease that we know of which localizes to lipid droplets, the lipid storage cell organelle. Previous studies have also shown that invadolysin mutants have a lower triglyceride to protein ratio and reduced fat body thickness and cross sectional area. Fat body in Drosophila is the functionally homolog of adipose tissue in higher organisms. Further suggesting a role of invadolysin in metabolism. In the Heck laboratory, invadolysin is studied using model organisms such as Drosophila melanogaster, Danio rerio (zebrafish) and cultured cell lines. During my PhD, my aim was to study the biosynthesis, activity and function of invadolysin and investigate its role in metabolism and adipogenesis. Invadolysin has a conserved metalloprotease motif ‘HEXXH’ and a potential lipase motif ‘GXSXS’. One of the aims of my PhD was to generate mutant versions of the conserved motifs to study their role on the activity of the proteins. I have generated transgenic flies that express wild type or E258A (protease dead) or S266A (lipase dead) versions of invadolysin. These transgenic flies would help in the study of the importance of the metalloprotease ‘HEILH’ and the lipase ‘GFSVS’ motifs in invadolysin’s activity. Transgenic flies overexpressing wild type and lipase dead form of invadolysin accumulate significantly higher levels of triglycerides as compared to control flies and transgenic flies overexpressing protease dead form of invadolysin. Suggesting a role of the protease motif in lipid accumulation. The other aim of my PhD was to study the role of invadolysin in metabolism. I followed up on previous observations in the laboratory that the insulin-signalling pathway is impaired in invadolysin mutant animals – with the hypothesis that invadolysin plays a role in metabolism and adipogenesis. I used Drosophila to study the effect on downstream targets of the insulin-signalling pathway such as triglyceride synthesis, glycogen synthesis and autophagy in invadolysin mutants. Results suggest that the insulin-signalling pathway and the ability to accumulate lipids are impaired in invadolysin mutants. Insulin also regulates adipogenesis by regulating the expression of PPARγ. I used SGBS cells, a human preadipocyte cell line to study the role of invadolysin in adipogenesis. Increase in protein levels of invadolysin during adipogenesis indicates a potential role of invadolysin in adipogenesis. Invadolysin has a predicted N-terminal signal sequence and also a predicted Cterminus GPI anchor site that suggests invadolysin can either be secreted or anchored to a membrane. Also, leishmanolysin, the closest homolog of invadolysin exists in a secreted and membrane bound form apart from a cytosolic form. This encouraged me to investigate the presence of a secreted form of invadolysin. Analysis of vertebrate and invertebrate plasma fractions of blood and hemolymph led to the identification of a novel secreted form of invadolysin. This novel discovery places invadolysin alongside a small group of metalloproteases, which are secreted into the extracellular environment and which play multiple roles in normal physiology and disease states.