Exploring the Structure and Dynamics of Ceramide Channels

University Of Maryland, College Park, College Park MD

Investigators

Abstract

Intellectual Merit: This project impacts three major areas: the role of lipids in cell function, the regulation of the initiation of apoptosis by mitochondria, and the self-assembly of nanostructures. The roles of lipids, in the context of membrane function, are generally relegated to two areas: the generation of a physical barrier between compartments and the regulation of the action of proteins. An additional role is the ability of ceramide, a sphingolipid, to form highly organized assemblies that generate large aqueous pores through phospholipid membranes. These channels allow proteins up to 60 kDa to cross membranes. This channel forming ability is limited to certain types of membranes: mitochondrial outer membranes, but not plasma membranes. This research aims to understand the structural basis underlying the ability of ceramide to form these large organized channels. A combination of experimental and computational (molecular dynamic simulations) approaches will be used to test the importance of various features of the ceramide molecule. The hypothesis is that the precise combination of a variety of features provides exactly the right shape and hydrogen-bonding ability to form the large channels. The presence of other sphingolipids that are either precursors or products in the ceramide metabolic pathway is expected to either promote or interfere with the assembly of these channels. Understanding these interactions is important in understanding the consequences of changes in the activity of the metabolic enzymes in this pathway and the consequent physiological changes. Among such physiological changes is the permeabilization of the mitochondrial outer membrane to proteins. This permeabilization is believed to release pro-apoptotic proteins that initiate the execution phase of apoptosis. Thus, understanding how ceramide channels form may allow one to find ways to control the initiation of apoptosis. The planned experiments will be performed both on phospholipid membranes, where the system is defined and free of proteins, and on isolated mitochondria. The former provides clear detailed mechanistic information, free of the complicating factors found in natural membranes. It also takes advantage of the high sensitivity and precision of the electrophysiological approach. The experiments on natural membranes provide the biological context, potentially revealing the presence of more complex interactions that would then be tested in the defined system. Molecular dynamic simulations will be used to test hypothetical structures and their dynamics in order to generate predictions to test experimentally. The use of simulated annealing will allow us to probe a variety of structures with limited computer time. Electron microscopy will be used to visualize the real structures for comparison with those inferred from function. The different approaches are expected to be synergistic and allow us to gain a solid understanding of the structures and their dynamics. Broader impact: This work will, in part, be performed by undergraduate students seeking to gain research experience and by exceptional high school students. These students will be under the direct supervision of at least one of the personnel supported by this grant. From the PI's prior experience, such selected students have proven to be highly motivated and they learn quickly. The students leave with greatly improved research skills and usually a new-found interest in continuing to be involved in research. Typically these students present the results of their research as posters in campus-wide competitions or as reports to high school classes. Thus the knowledge and experience is shared with others. Typically the PI's lab has a great diversity of students, including many minority students. The PI often shares insight gained in the research lab with students in his upper-level classes. The molecular dynamics simulation will be a collaboration between the laboratory of the PI and that of Sergei Sukharev whose post-doctoral fellow is will be supported by this award. The modeling facility is primarily used to study the structure and dynamics of stretch-sensitive channels. As a result of this collaboration, new molecular dynamics methods are being developed. This is the only molecular dynamics simulation facility on campus and the planned research will expand its utility and visibility. In terms of potential benefits to society, understanding the structural features that allow ceramide to form these large channels may have implications on the study of the self-assembly of nanostructures and lead to practical applications. For example, this knowledge could be used to help design liposome systems (e.g., for delivery of contents to specific sites) that could be regulated in terms of the loading and unloading of their cargoes.