Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. Furthermore, we understand even less about how the major HDL protein, apolipoprotein (apo)A-I, interacts with other proteins to dictate HDL function. This project will test the hypothesis that apoA-I makes highly specific contacts with it and other HDL associated proteins in spherical HDL particles commonly found in human plasma. In our previous work, we used cross- linking chemistry and mass spectrometry to generate detailed models of apoA-I in reconstituted particles as well as """"""""real"""""""" HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt-like arrangement. Building on these discoveries, our goal is to further evaluate these and other models using complementary structural techniques as well as evaluate the basis of apoA-I's interactions with three major HDL components: apolipoprotein A-II, lecithin:cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein (CETP).
The specific aims are: 1) To test the Trefoil-based models of apoA-I in spherical reconstituted and native plasma HDL using new dual isotope cross-linking techniques and state-of-the-art all-atom and course grained molecular dynamics (MD) techniques. 2) To determine the molecular interactions between apoA-I and apoA-II using cross-linking and a new human apoA-II bacterial expression system to derive the first models of native HDL particles containing both proteins. 3) To determine the molecular interaction between apoA-I and LCAT by testing the concept that LCAT accesses a molecular pore in the apoA-I structure using site-directed mutagenesis and cross-linking. We will also determine if CETP specifically binds apoA-I and identify their points o contact. This approach uniquely intertwines new experimental techniques with state-of-the-art MD approaches that will magnify our structural understanding of these enigmatic particles. The structure of apoA-I undoubtedly modulates HDL metabolism. Thus, a molecular understanding of its structure and its interactions with other proteins, particularly those being explored as dru targets such as LCAT and CETP, is critical for the design of new therapies exploiting reverse cholesterol transport and the anti-inflammatory roles of HDL.

Public Health Relevance

The proposed research is relevant to public health because an increased understanding of the function of high density lipoproteins (HDL) will help guide the development of therapeutic strategies designed to raise plasma HDL for protection against cardiovascular disease, the # 1 killer in the U.S. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge for understanding the causes, prevention and eventually a cure for human diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL067093-10
Application #
8504414
Study Section
Special Emphasis Panel (ZRG1-EMNR-P (02))
Program Officer
Liu, Lijuan
Project Start
2001-04-01
Project End
2017-05-31
Budget Start
2013-07-05
Budget End
2014-05-31
Support Year
10
Fiscal Year
2013
Total Cost
$379,931
Indirect Cost
$123,465
Name
University of Cincinnati
Department
Pathology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
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Davidson, W Sean (2014) HDL-C vs HDL-P: how changing one letter could make a difference in understanding the role of high-density lipoprotein in disease. Clin Chem 60:e1-3
Walker, Ryan G; Deng, Xiaodi; Melchior, John T et al. (2014) The structure of human apolipoprotein A-IV as revealed by stable isotope-assisted cross-linking, molecular dynamics, and small angle x-ray scattering. J Biol Chem 289:5596-608

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