Robert O. RyanAdjunct Professor Senior Scientist at Children's Hospital Oakland Research Institute (510) 450-7645 |
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Function of Exchangeable Apolipoproteins
Heart disease is a major cause of mortality in North America. While it is clear that development of cardiovascular disease is a multi-factorial process, it is evident that aberrations in lipid metabolism represent a significant risk factor. It is widely accepted that exchangeable apolipoproteins function in regulation of plasma lipid levels, yet the molecular basis for this role is not fully understood. Increased knowledge of the properties of exchangeable apolipoproteins will be useful in the development of therapeutic strategies to influence plasma lipid levels and thereby, reduce the risk of cardiovascular disease. Furthermore, by understanding the molecular basis of apolipoprotein function in lipid transport and metabolism, it should be possible to design strategies to enhance or interfere with biological processes dependent upon their action. Knowledge gained has direct relevance to the treatment of dyslipidemias, including hypercholesterolemia and disorders of lipid metabolism that affect children and adults.
Ryan Research Program Summary
The laboratory has three main research projects that are focused on a family of plasma proteins that function to regulate lipid transport and metabolism.
In project 1 the goal is to understand how lipid transport and metabolism are regulated by molecular interactions between lipoproteins and cell surface receptors. The interaction of apolipoprotein (apo) E with the low-density lipoprotein receptor (LDLR) is under investigation to identify molecular determinants required for a productive receptor-ligand interaction. We have shown that apoE undergoes a lipid binding induced conformational change that results in extension of helix 4 beyond the boundary identified in its lipid-free helix bundle state. More recently we have employed Expressed Protein Ligation to reconstruct an intact apoE molecule from fragments generated in a bacterial expression system. This approach has permitted specific isotope enrichment of only a portion of the apoE molecule, facilitating detailed structural analysis. At the same time, a soluble fragment of LDLR has been employed in ligand binding and release studies with apoE. LDL-A repeat swapping experiments and modification of the spacer sequences between repeats is under study to determine the molecular requirements for a productive interaction with apoE containing reconstituted high density lipoprotein ligands. These studies will reveal new insight into the mechanism whereby plasma cholesterol levels are regulated.
In project 2 we are exploring the mechanism whereby a recently discovered plasma apolipoprotein, apoA-V, modulates plasma triacylglycerol levels. Structure-function and site directed mutagenesis experiments have been used to show that apoA-V interacts with heparan sulfate proteglycans (HSPG). Current research efforts are designed to test the hypothesis that apoA-V functions by facilitating interaction of very low-density lipoproteins (VLDL) with HSPGs, thereby enhancing lipoprotein lipase mediated lipolysis of VLDL associated triacylglycerol. In other studies, we have shown that apoA-V associates with cytosolic lipid droplets and is comprised of two structural domains, an N-terminal helix bundle motif and a C-terminal lipid-binding segment. Current studies employed transgenic and gene disrupted mice are designed to delineate the structural basis for the triacylglycerol lowering effects of apoA-V.
In project 3 we are investigating the ability of recombinant human apoA-I to solubilize phospholipid dispersions, generating a homogeneous population of water-soluble, nanometer scale lipid particles, termed nanodisks. One goal of this research is to incorporate hydrophobic biomolecules and employ the resulting particles as water-soluble transport vehicles. Recent success incorporating the polyene antibiotic amphotericin B and all trans retinoic acid illustrate the potential utility of these particles. Cell culture and in vivo experiments in mice have revealed that nanodisk-associated biomolecules retain their biological activity and can be targeted to cell surface receptors via their intrinsically associated apolipoprotein component.
Most Recent Publications
Lookene, A., Beckstead, J.A., Nilsson, S., Olivecrona, G. and Ryan, R.O. (2005) Apolipoprotein A-V heparin interactions. Implications for plasma lipoprotein metabolism. J. Biol. Chem. 280, 25383-25387.
Gupta, V., Narayanaswami, V., Budamagunta, M.S., Yamamoto, T., Voss, J.C. and Ryan, R.O. (2006) Lipid induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability. J. Biol. Chem. 281, 39294-39299.
Nilsson, S.K., Lookene, A., Beckstead, J.A., Gliemann, G. Ryan, R.O. and Olivecrona, G. (2007) Apolipoprotein A-V interaction with members of the Low Density Lipoprotein Receptor Gene Family. Biochemistry, 46, 3896-3904.
Beckstead, J.A. Wong, K., Gupta, V., Wan, C.-P. L., Cook, V.R., Weinberg, R.B., Weers, P.M.M., and Ryan, R.O. (2007) The effect of C-terminal truncation on the structural and lipid binding properties of apolipoprotein A-V. J. Biol. Chem. 282, 15484-15489.
Wong, K., Lee, D., Beckstead, J.A., Weers, P.M.M., Kay, C.M. and Ryan, R.O. (2008) The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture. Biochemistry. Epub ahead of print.