Presentation Title

Spectroscopic and Mass Spectrometric Analysis of ApoAI during HDL Particle Modulation by Arterial Endothelial Cells

Faculty Mentor

Dr. Vasanthy Narayanaswami

Start Date

23-11-2019 9:00 AM

End Date

23-11-2019 9:15 AM

Location

Markstein 102

Session

oral 1

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

The last decade has seen a paradigm shift in the concept that the functionality of high density lipoproteins-cholesterol (HDL-C) may be more important than its overall level within the body as a determining factor in cardiovascular disease (CVD). Numerous studies show evidence that neither pharmacological nor genetic interventions to increase HDL-C levels lowered the risk for CVD. Thus, there is a need to understand the role of HDL in CVD from a mechanistic perspective and understand structure-function relationships in the atheroprotective property of HDL. HDL are large lipid-protein complexes with apolipoprotein (apo) AI being an important component in cholesterol transport. The overarching goal of this project is to understand conformational reorganization of apoAI and HDL during cholesterol efflux and HDL transcytosis across the arterial intima endothelium. We will test the hypothesis that apoAI folds in on itself at residues 125-158, when less than maximally engorged with lipids. To address this issue three double-cysteine mutants of apoAI were over-expressed in E. coli and purified using affinity chromatography. The cysteines were labeled with N-(1-pyrene)-maleimide (pyrene), yielding a labeling stoichiometry of ~2:1 (pyrene: apoAI). Pyrene is a spatially-sensitive probe that exhibits a unique spectral feature called ‘excimer’ at ~460 nm when proximal to a neighboring pyrene at ~ 10 Å. Fluorescence spectroscopic analysis indicated robust labeling of all the apoAI variants. The protein will be complexed with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine by the cholate dialysis method to yield reconstituted HDL particles. Subsequently, bovine aortic endothelial cells will be exposed to pyrene-labeled apoAI variants in lipid-free and rHDL forms, and pyrene fluorescence emission spectra will be recorded under various conditions. The conditioned medium from cells exposed to labeled apoAI will be examined for lipid acquisition of apoAI by lipid analysis, agarose and native PAGE to assess size, and lipidomic analysis to obtain a profile of the lipids.

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Nov 23rd, 9:00 AM Nov 23rd, 9:15 AM

Spectroscopic and Mass Spectrometric Analysis of ApoAI during HDL Particle Modulation by Arterial Endothelial Cells

Markstein 102

The last decade has seen a paradigm shift in the concept that the functionality of high density lipoproteins-cholesterol (HDL-C) may be more important than its overall level within the body as a determining factor in cardiovascular disease (CVD). Numerous studies show evidence that neither pharmacological nor genetic interventions to increase HDL-C levels lowered the risk for CVD. Thus, there is a need to understand the role of HDL in CVD from a mechanistic perspective and understand structure-function relationships in the atheroprotective property of HDL. HDL are large lipid-protein complexes with apolipoprotein (apo) AI being an important component in cholesterol transport. The overarching goal of this project is to understand conformational reorganization of apoAI and HDL during cholesterol efflux and HDL transcytosis across the arterial intima endothelium. We will test the hypothesis that apoAI folds in on itself at residues 125-158, when less than maximally engorged with lipids. To address this issue three double-cysteine mutants of apoAI were over-expressed in E. coli and purified using affinity chromatography. The cysteines were labeled with N-(1-pyrene)-maleimide (pyrene), yielding a labeling stoichiometry of ~2:1 (pyrene: apoAI). Pyrene is a spatially-sensitive probe that exhibits a unique spectral feature called ‘excimer’ at ~460 nm when proximal to a neighboring pyrene at ~ 10 Å. Fluorescence spectroscopic analysis indicated robust labeling of all the apoAI variants. The protein will be complexed with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine by the cholate dialysis method to yield reconstituted HDL particles. Subsequently, bovine aortic endothelial cells will be exposed to pyrene-labeled apoAI variants in lipid-free and rHDL forms, and pyrene fluorescence emission spectra will be recorded under various conditions. The conditioned medium from cells exposed to labeled apoAI will be examined for lipid acquisition of apoAI by lipid analysis, agarose and native PAGE to assess size, and lipidomic analysis to obtain a profile of the lipids.