Defining the Roles of Various Lysines and Arginines in Amot Lipid Binding

Abstract

One of the defining traits of cancerous cells is proliferation. We are focusing on the proliferation of mammary cells. As an adaptor protein, the Amot membrane binding event is key to the localization and sorting of proteins responsible for cellular differentiation, proliferation, and migration. The Amot coiled-coil homology domain (ACCH) is a lipid-binding domain responsible for affinity and binding to endothelial membranes. Our working hypothesis is that the ability to modulate Amot lipid-binding will lead to means to prevent ductal cell hyperplasia progression into breast cancer tumors. We will determine which residues are responsible for lipid-binding by changing positively charged lysine and arginine into uncharged or negatively charged amino acids. Those mutations which show a significant decrease in lipid-binding will then be used to determine their down-stream effects in human cells. The laboratory has screened approximately 40 of these mutations using a liposome binding assay. This assay mimics how the protein binds with the cell membrane by using an in vitro mixture of lipids similar to that seen in endothelial cells. Forster resonance energy transfer (FRET) was used to confirm significant decreases in lipid binding of ACCH mutants selected from the liposome binding assay, as energy transfer only occurs when the tyrosines in the protein and the Dansylated liposome are in close proximity to each other. Based on these two screens we have narrowed the list to seven mutants that have a significant decrease in lipid binding. Currently, FRET is being used to determine the lipid binding coefficient for each mutant of interest. Mutants deemed important from this study will then be transformed into human cells to study their effects on cell polarity, signal transduction, cell shape, and cellular proliferation.