The overall objective is to gain a more complete understanding of the structure of apolipoprotein (apo) E, especially as it relates to the ability of the protein to bind to the low density lipoprotein (LDL) receptor and lipids. Point mutations are known to give isoforms of apo E that function abnormally in cholesterol and triglyceride transport. A range of physical-biochemical techniques will be used with apo E engineered and expressed in E. coli to address 3 specific aims. 1) To understand how interaction with phospholipid (PL) changes the conformation of apo E3 so that it can bind to the LDL receptor. The microenvironments of the lysine (K) residues in lipoprotein-associated, amphipathic, a-helices reflect interactions between helices so the pKa of each 13C-labeled K residue in PL-protein discoidal particles will be determined by (1H, 13C)-heteronuclear multiple quantum coherence NMR. The hypothesis to be tested is that the a-helix lengths and spacings induced by interaction with the lipid are critical for achieving high affinity binding to the LDL receptor. 2) To understand the mechanisms responsible for the differing affinities of apo E isoforms for variously-sized serum lipoprotein particles, the thermodynamic parameters characterizing the binding of apo E and engineered variants to a range of lipid particles of defined sizes will be measured using fluorescence spectroscopy and titration calorimetry. 3) To understand how the microrenvironment in the LDL receptor- binding domain of the apo E molecule correlates with the energetics of binding, the microenvironments of K143 and K146 which are directly involved in receptor binding will be investigated using apo E in states where its free energy of binding to the receptor varies widely. Changes in the receptor binding domain will be induced by either mutagenesis of the apo E molecule or changes in the associated lipid. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apo E structure and polymorphism affect the functional properties of the protein in both physiological and pathological conditions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056083-04
Application #
2857873
Study Section
Metabolism Study Section (MET)
Project Start
1997-01-01
Project End
2000-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Mcp Hahnemann University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19102
Mizuguchi, Chiharu; Hata, Mami; Dhanasekaran, Padmaja et al. (2014) Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4. Biochim Biophys Acta 1841:1716-24
Nguyen, David; Dhanasekaran, Padmaja; Nickel, Margaret et al. (2014) Influence of domain stability on the properties of human apolipoprotein E3 and E4 and mouse apolipoprotein E. Biochemistry 53:4025-33
Li, Hui; Dhanasekaran, Padmaja; Alexander, Eric T et al. (2013) Molecular mechanisms responsible for the differential effects of apoE3 and apoE4 on plasma lipoprotein-cholesterol levels. Arterioscler Thromb Vasc Biol 33:687-93
Kothapalli, Devashish; Castagnino, Paola; Rader, Daniel J et al. (2013) Apolipoprotein E-mediated cell cycle arrest linked to p27 and the Cox2-dependent repression of miR221/222. Atherosclerosis 227:65-71
Phillips, Michael C (2013) New insights into the determination of HDL structure by apolipoproteins: Thematic review series: high density lipoprotein structure, function, and metabolism. J Lipid Res 54:2034-48
Mizuguchi, Chiharu; Hata, Mami; Dhanasekaran, Padmaja et al. (2012) Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4. Biochemistry 51:5580-8
Kothapalli, Devashish; Liu, Shu-Lin; Bae, Yong Ho et al. (2012) Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression and arterial stiffening. Cell Rep 2:1259-71
Nguyen, David; Dhanasekaran, Padmaja; Nickel, Margaret et al. (2010) Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4. Biochemistry 49:10881-9
Lund-Katz, Sissel; Phillips, Michael C (2010) High density lipoprotein structure-function and role in reverse cholesterol transport. Subcell Biochem 51:183-227
Koyama, Mao; Tanaka, Masafumi; Dhanasekaran, Padmaja et al. (2009) Interaction between the N- and C-terminal domains modulates the stability and lipid binding of apolipoprotein A-I. Biochemistry 48:2529-37

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