2019-2020 Grant in Aid of Research
Exploring the Role of Carboxypeptidase O and Its Role in Chylomicron and Lipid Droplet Formation
Erika Bauza Nowotny
Carboxypeptidase O (CPO) cleaves acidic and polar C-terminal amino acids of peptides and proteins; however, its biological function remains unknown. CPO is expressed in enterocytes in human intestinal tissue. At the subcellular level, CPO is anchored to the inner leaflet of the endoplasmic reticulum (ER) membrane and associated with lipid droplets. Additionally, CPO plays a role in lipid droplet formation in Madin-Darby Canine Kidney (MDCK) cells. Due to the role of the intestine in the formation of chylomicrons through similar mechanisms, it is appropriate to investigate a potential role of CPO in formation of chylomicrons, which bud off from the inner leaflet of the ER membrane into the lumen. Here, I propose to stably express CPO in an enterocyte-like cell line, human colon carcinoma (Caco-2) cells, and to test the localization and association of CPO with lipids within the ER of these cells. Cells expressing wild type CPO and inactive CPO mutants will be stained to observe potential differences in the amount of lipid droplets and chylomicrons that associate with CPO. Additionally, I will perform immunoprecipitation of wild type and inactive CPO from cell lysates and identify potential binding partners and substrates by tandem mass spectrometry (MS/MS). Ultimately, this identification will give me information about the intracellular function of CPO. It is possible that my experimental results may reveal that the major binding partner or substrate of CPO is a protein known to be associated with lipid droplet or chylomicron formation.