Protein Sensors for Membrane Cholesterol

The cholesterol content of animal cells is maintained within narrow limits. This regulation applies not only to the overall cholesterol level in the cell, but also to the concentration of cholesterol in the membrane of each organelle. Maintaining the proper level and distribution of cholesterol, which is virtually insoluble, requires an efficient network of proteins that can measure cholesterol levels in membranes and transport cholesterol. Despite much interest, our understanding of how cholesterol sensors accurately measure cholesterol levels in membranes and how cholesterol is transferred between membranes remains limited. A recently discovered class of cholesterol-sensing bacterial toxins share the same specificity and sensitivity for cholesterol as mammalian cholesterol sensors. Using two members of this large family, perfringolysin O and anthrolysin O, I showed that sigmoidal responses of cholesterol sensors can arise primarily from membrane effects due to sharp changes in the chemical activity of cholesterol. The nonlinear response emerges because interactions between bilayer lipids control cholesterol accessibility to sensors in a threshold-like fashion. Around these thresholds, the affinity of sensors for membrane cholesterol varies by >100-fold, generating highly cooperative lipid-dependent responses independently of protein-protein interactions. I then used supported bilayer technology and fluorescently labeled anthrolysin O to devise an ultrasensitive method to measure protein-mediated cholesterol transport between fluid membranes. Using this method, I showed that human Niemann Pick Type C2 disease protein and yeast oxysterol-binding protein homolog transport cholesterol between membranes. I also showed that two point mutations in Niemann Pick Type C2 disease protein that cause cholesterol storage disease also render the protein defective in transporting cholesterol between membranes. I then characterized the lipid binding properties of a fungal toxin ostreolysin A, and showed that it binds membranes only when they contain cholesterol and sphingomyelin. I developed fluorescently labeled ostreolysin A as a tool to probe for sphingomyelin-cholesterol complexes in the plasma membranes of mammalian cells. In ongoing work, I am screening for compounds that bind anthrolysin O, as candidates for inhibitors of the human cholesterol sensor, Scap. The tools that I have developed will improve our understanding of cholesterol regulation in animal cells.