Members of the TMEM16 family of integral membrane proteins are Ca2+-dependent phospholipid scramblases. Because mechanisms of lipid scrambling by the TMEM16s remain poorly understood, the ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate the activity of these proteins, is limited. Our goal is to overcome these limitations by determining how the TMEM16 scramblases are activated in response to Ca2+ binding, how and for what purpose they remodel cellular membranes, and how they are affected by specific components of these membranes. We address this mechanistic goal with an integrated strategy combining experimentation with structural, functional, and computational approaches. To understand how these proteins are modulated in vivo we will focus on ceramides as the first class of molecules found to inhibit the function of TMEM16 scramblases and to be associated in vivo with excessive exposure of PS in endothelial cells. Our 1st aim is to determine the Ca2+- dependent gating mechanism of the TMEM16 scramblases using a combination of cryo-electron microscopy (cryoEM), molecular dynamics (MD) simulations and functional assays. These experiments will reveal the allosteric coupling mechanism between the Ca2+ binding site and the structural elements gating the lipid pathway. Our 2nd aim is to determine how the TMEM16 scramblases interact with, and alter the structure of, their surrounding membrane environment in support of their function. Using structure determination with cryoEM we will visualize afTMEM16 complexes with membranes with a variety of physicochemical properties and compositions, in different functional states. In combination with MD simulations and functional assays we will identify the energetic and molecular determinants for membrane-protein interactions and membrane remodeling, and their role in scrambling. Our 3rd aim is to determine the mechanism and in vivo role of ceramide regulation of TMEM16 scramblases using functional assays to identify the molecular determinants of ceramide inhibition, and structural and computational experiments to determine their mechanism of action, and the role of specific ceramides in the in vivo regulation of TMEM16F.
The TMEM16-type scramblases perform important functions in the cell by mediating the exposure of the negatively charged lipid phosphatidylserine in the leaflet of the plasma membrane that faces the cell's external environment, a key trigger in a variety of signaling processes such as coagulation of blood cells, clearance of dying (apoptotic) cells, membrane repair and fusion. Despite recent progress in elucidating the structure of TMEM16 molecules, our ability to recognize their functions in human physiology and to design targeted pharmacological interventions is limited by our poor understanding of the mechanisms that make possible the entry of the moved (scrambled) lipids (termed gating) and of the and modulation of these proteins. Therefore, the goal of our project is to overcome these limitations by determining how the dynamic rearrangements of the TMEM16 scramblases in response to Ca2+ binding enable their function, how they alter the structure of cellular membranes and, vice versa, how specific components of these membranes modulate their activity.
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|Picollo, Alessandra; Malvezzi, Mattia; Accardi, Alessio (2015) TMEM16 proteins: unknown structure and confusing functions. J Mol Biol 427:94-105|