Characterization of Invadolysin in flies and human cells: new insights into a novel, essential metalloprotease

Metalloproteases are enzymes that cleave other proteins and require a metal ion fo r their activity. They play a major role in the regulation of several cellular processes. I have analysed the functions of a novel, conserved metalloprotease named Invadolysin, previously reported by the Heck laboratory to be involved in mitosis and cell migration.I carried out a second site non-complementation screen in Drosophila melanogaster to identify the interactors of lnvadolysin utilizing the availability of a collection of genome-wide deficiency stocks. As a result of this screen, I identified non-stop, a ubiquitin protease, as a genetic interactor of invadolysin. I characterized similar mutant phenotypes of invadolysin and non-stop and showed that the abnormal chromosomal architecture observed in both mutants might be a result of histone ubiquitination.I also investigated the involvement of lnvadolysin in Notch pathway. The Notch extracellular domain levels were normal in the invadolysin mutants, but the intracellular domain levels were greatly reduced suggesting that the Notch pathway was inactive, or compromised. The levels of the Notch ligand Delta were abnormally high in invadolysin mutants, suggesting a block of Notch activity through cis-inactivation due to a possible failure in endocytosis of the Delta ligand. In human cells, I showed that Invadolysin partially co-localized with Rab II and Itch proteins, which have been shown to influence the Notch pathway.Considering the possible involvement of Invadolysin in Delta endocytosis, I examined the colocalization of lnvadolysin with endocytosis machinery in human cells. I found that lnvadolysin rings partially localized with Caveolin-1, and the caveolar endocytosis marker cholera toxin B was found inside the Invadolysin ring-like structures, suggesting that the inner region of Jnvadolysin rings might be a lipid based entity. When the lipid droplet marker BODIPY was utilized with Invadolysin, I established that Invadolysin surrounds lipid droplets.Together, these studies have identified a previously unknown involvement of Invadolysin in the regulation of chromosome architecture, and the Notch pathway. The localization of Invadolysin to lipid droplets suggests its site of action and provides novel avenues to the study of this intriguing and essential metalloprotease