One of the defining traits of cancerous cells is proliferation. The adaptor protein, Amot has a membrane binding event 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 phosphoinositol and cholesterol affinity which lend to binding epithelial 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. Approximately 40 mutations of lysines and arginines into uncharged or negatively charged amino acids have been screened using a liposome binding assay which mimics how the protein binds with the cell membrane by using an in vitro mixture of lipids similar to that seen in endothelial cells. Mutations were screened for significant decreases in phosphoinositol liposome binding with Forster resonance energy transfer (FRET) and lipid sedimentation, and we have selected 7 mutants for further study. Previous work suggests Amot binding affinity is increased in the presence of cholesterol. To further our understanding of the role of cholesterol in membrane association, we studied the effect of cholesterol concentrations on binding affinity for each of these mutants. This study provides information on the mechanisms involved in the protein membrane binding events involved in membrane polarity and signal transduction.

Defining the roles of various lysines and arginines in Amot lipid binding

247th National Meeting (2014)

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