Description The long-term goal of our research is to understand how molecular interactions between lipoproteins and cell surface receptors affect lipid metabolism and disease susceptibility. Knowledge of the metabolic roles of apolipoprotein (apo) E and low-density lipoprotein receptor (LDLR) family members will be combined with available structural information in the design of an experimental strategy to dissect determinants of a productive binding interaction. A novel strategy, termed expressed protein ligation, will be employed to introduce stable isotopes into a specific, predetermined, region of apoE that is essential for LDLR binding. The research to be pursued includes three specific aims. 1) Segmental isotope labeled apoE3-N-temrinal domain, in complex with lipid, will be analyzed by multidimensional heteronuclear NMR spectroscopy in experiments designed to distinguish between alternate structural models of the lipid-bound apoE. 2) The contact sites between apoE and the LDLR will be characterized. It is hypothesized that analysis of segmental isotope labeled apoE3-NT7DMPC in complex with a functional LDLR mini-receptor will yield molecular details of their binding interaction. 3) The interaction of apoE with LDL receptor related protein 6 (LRP6) will be studied. The hypothesis that LDL-A repeats located near the transmembrane spanning sequence of LRP6 are involved in apoE ligand binding will be evaluated. In addition, the postulate that a mutation in LRP6 (R611C), associated with coronary artery disease in human subjects, causes a defect in pH dependent apoE ligand release will be tested. The results of these experiments will extend knowledge of the LDLR family, apoE-mediated lipoprotein metabolism and regulation of plasma lipid homeostasis. Based on the fact that aberrations in the LDLR pathway are positively correlated to onset of cardiovascular disease and apoE manifests isoform-specific susceptibility to neurodegenerative diseases, we anticipate that new knowledge gained from these studies will provide insight into molecular mechanisms that regulate key metabolic processes in health and disease.

Public Health Relevance

Apolipoprotein (apo) E is an important modulator of whole body lipid homeostasis. Biological functions of apoE are manifest through interactions with a family of cell surface receptors. Proposed research will characterize the receptor-active structure of apoE as well as its binding interaction with two members of the low-density lipoprotein receptor family. Results obtained will improve understanding of how apoE regulates lipid metabolism and its association with disease susceptibility.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL064159-15
Application #
8668125
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Liu, Lijuan
Project Start
2000-06-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital & Res Ctr at Oakland
Department
Type
DUNS #
City
Oakland
State
CA
Country
United States
Zip Code
94609
Krishnamoorthy, Aparna; Witkowski, Andrzej; Tran, Jesse J et al. (2017) Characterization of secondary structure and lipid binding behavior of N-terminal saposin like subdomain of human Wnt3a. Arch Biochem Biophys 630:38-46
Ikon, Nikita; Shearer, Jennifer; Liu, Jianfang et al. (2017) A facile method for isolation of recombinant human apolipoprotein A-I from E. coli. Protein Expr Purif 134:18-24
Krishnamoorthy, A; Witkowski, A; Ryan, R O (2017) Nutlin-3a Nanodisks Induce p53 Stabilization and Apoptosis in a Subset of Cultured Glioblastoma Cells. J Nanomed Nanotechnol 8:
Ikon, Nikita; Ryan, Robert O (2017) Cardiolipin and mitochondrial cristae organization. Biochim Biophys Acta 1859:1156-1163
Ikon, Nikita; Ryan, Robert O (2017) Barth Syndrome: Connecting Cardiolipin to Cardiomyopathy. Lipids 52:99-108
Forte, Trudy M; Sharma, Vineeta; Ryan, Robert O (2016) Apolipoprotein A-V gene therapy for disease prevention / treatment: a critical analysis. J Biomed Res 30:88-93
Ikon, Nikita; Ryan, Robert O (2016) On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism. J Inherit Metab Dis 39:749-756
Lalefar, Nahal R; Witkowski, Andrzej; Simonsen, Jens B et al. (2016) Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells. J Nanobiotechnology 14:66
Crosby, Natasha M; Ghosh, Mistuni; Su, Betty et al. (2015) Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas. Biochem Cell Biol 93:343-50
Ikon, Nikita; Su, Betty; Hsu, Fong-Fu et al. (2015) Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells. Biochem Biophys Res Commun 464:580-5

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