Despite the strong relationship of elevated plasma levels of high density lipoproteins (HDL) with protection from cardiovascular disease (CVD), the molecular details of how newly secreted lipid-free apolipoproteins transition to lipidated HDL particles remains unknown. This gap in understanding is a critical hindrance as the pharmaceutical industry is in the midst of a major effort to identify therapies that raise plasma HDL levels. Our long term goal is to understand how HDL is generated and whether this process can be manipulated for therapeutic benefit. Pursuant to this, our objective here is to derive a molecular understanding of the lipid binding structural transitions of two prototypical HDL apolipoproteins: apolipoprotein (apo)A-I and apoA-IV. Since these proteins interact with lipid in distinct ways, we aim to define a range of mechanistic themes that will be applicable to the broader apolipoprotein family. Our central hypothesis is that apolipoprotein self-association is a critical feature that dictates a given protein's ability to generate HDL. This is based on the fact that apolipoproteins universally self-associate as well as our preliminary data showing a clear and highly novel mechanism for how they accomplish this. Our rationale is that understanding apolipoprotein structural adaptations during lipid binding will provide new insights into the mechanism of HDL formation in vivo. To test our hypothesis, we will pursue three specific aims: 1) Determine the structure and self-association mechanism of lipid-free apoA-I and apoA-IV;2) Determine the structure of apoA-I and apoA-IV in their lipid-bound states;and 3) Determine the importance of self-association and test molecular folding pathways for apoA-I and apoA- IV lipoprotein particle assembly.
In Aims 1 and 2 we will derive badly needed high-resolution structures of these apolipoproteins at the beginning and end of the HDL particle assembly process by X-ray crystallography, complemented by solution-based spectroscopic and cross-linking experiments. This knowledge will be used in Aim 3 to alter apoA-I and apoA-IV self-association properties and test the effects on ability to generate HDL particles either by spontaneous lipid reorganization or via cell-based HDL particle formation assays. We will also introduce disulfide constraints to test detailed schemes for the lipid-binding structural transitions undertaken by both proteins. This work is innovative because we have discovered a novel """"""""helix swapping"""""""" mechanism that explains apoA-IV self-association and likely other apolipoproteins as well. This provides a new conceptual framework upon which a better understanding of apolipoprotein function can be built and it offers a clear basis for the arrangements that apolipoproteins adopt in HDL particles. Finally, the work is significant because it will illuminate the molecular events that occur during HDL formation as well as mechanisms for dissociation of apolipoproteins from pre-formed HDL particles, both critical processes governing HDL metabolism. This understanding will help guide the development of therapeutic strategies designed to raise plasma HDL for protection against CVD, the number 1 killer in the U.S.

Public Health Relevance

The proposed research is relevant to public health because an increased understanding of the biogenesis 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 Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098458-03
Application #
8499379
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Chin, Jean
Project Start
2011-09-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$287,860
Indirect Cost
$104,510
Name
University of Cincinnati
Department
Pathology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Melchior, John T; Walker, Ryan G; Cooke, Allison L et al. (2017) A consensus model of human apolipoprotein A-I in its monomeric and lipid-free state. Nat Struct Mol Biol 24:1093-1099
Melchior, John T; Street, Scott E; Andraski, Allison B et al. (2017) Apolipoprotein A-II alters the proteome of human lipoproteins and enhances cholesterol efflux from ABCA1. J Lipid Res 58:1374-1385
Melchior, John T; Walker, Ryan G; Morris, Jamie et al. (2016) An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding. J Biol Chem 291:5439-51
Gordon, Scott M; Li, Hailong; Zhu, Xiaoting et al. (2015) A comparison of the mouse and human lipoproteome: suitability of the mouse model for studies of human lipoproteins. J Proteome Res 14:2686-95
Deng, Xiaodi; Walker, Ryan G; Morris, Jamie et al. (2015) Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function. J Biol Chem 290:10689-702
Lo, Chunmin C; Davidson, W Sean; Hibbard, Stephanie K et al. (2014) Intraperitoneal CCK and fourth-intraventricular Apo AIV require both peripheral and NTS CCK1R to reduce food intake in male rats. Endocrinology 155:1700-7
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
Deng, Xiaodi; Morris, Jamie; Chaton, Catherine et al. (2013) Small-angle X-ray scattering of apolipoprotein A-IV reveals the importance of its termini for structural stability. J Biol Chem 288:4854-66
Deng, Xiaodi; Morris, Jamie; Dressmen, James et al. (2012) The structure of dimeric apolipoprotein A-IV and its mechanism of self-association. Structure 20:767-79
Deng, Xiaodi; Davidson, W Sean; Thompson, Thomas B (2012) Improving the diffraction of apoA-IV crystals through extreme dehydration. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:105-10