STRUCTURAL AND CELL BIOLOGY IN CARDIOVASCULAR DISEASE

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The overall objective of this program is to understand the Structural Biology and hence, function of the macromolecules and macromolecular complexes involved in the transport of lipids into and out of cells. The aims of the projects are: to define the 3-dimensional structures of cellular receptors and their ligands, such as the LDL receptor bound to the LDL Project; to study the formation of primordial nascent triglyceride-rich particles in the endoplasmic reticulum and understand how the secondary and tertiary structure of the N-terminal 41% apo-B regulates this process Project; to understand the structure and conformation of apo-B, the 3-dimensional structure of LDL, the organization of apo-B on LDL, and to define the conformation and 3- dimensional structure of exchangeable apolipoproteins on nascent and plasma HDL Project; to understand the 3-dimensional structure of albumin and its specific fatty acid-binding domains, the transfer of fatty acids from albumin and movement across membranes into cells, their consequent interaction with fatty acid binding proteins, and the effect of fatty acids on intracellular pH and calcium Project. State-of-the-art techniques of structural biology are used to study isolated or reconstituted macromolecular complexes, consisting of proteins or lipid- protein complexes and aggregates. Low resolution structures of individual particles (and/or 2-dimensional arrays) are obtained by electron microscopy, in particular cryo-EM, combined with image analysis and reconstruction. Decorating these structures with site-specific labels, such as antibodies to specific epitopes, gold bound to free cysteine, antibody/Fabs, ligand, etc., aids in defining the position in specific domains or sequences in the macromolecular complex. To determine detailed structures at molecular resolution, individual proteins or protein-lipid complexes are crystallized and the structure determined by x-ray crystallography. The solution structure of peptides that model specific regions of proteins or small proteins such as the intracellular fatty acid binding proteins are determined by multi-dimensional NMR. High resolution molecular arrangements can be superimposed onto the low resolution structure of macromolecular assemblies obtained by electron microscopy to generate a "higher resolution" macromolecular structure. These studies will provide fundamental molecular information essential to understanding the biological structures involved in the processes by which lipids are moved into, within, and out of cells. Such information will allow the development of new molecular-based strategies to control hyper beta lipoproteinemia, fatty acid-induced cellular damage in ischemia, and arteriosclerosis.