The Impact of Cigarette Smoking on HDL Composition and Function Katie Thomas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2014 Reading Committee: Jay Heinecke, Chair Karin Bornfeldt Daniel Bowen-Pope Program Authorized to Offer Degree: Molecular and Cellular Biology Copyright 2014 Katie Thomas University of Washington Abstract The Impact of Cigarette Smoking on HDL Composition and Function Katie Thomas Chair of the Supervisory Committee: Jay Heinecke, MD Department of Medicine It has been known for decades that cigarette smoking is associated with atherosclerosis; however, the specific mechanisms for this association remain unclear. High density lipoprotein (HDL) is known to play a key atheroprotective function by removing excess cellular cholesterol, thereby reversing atherosclerotic lesion formation. Here, we investigate whether smoking causes oxidation of HDL proteins, leading to loss of function. We utilized three separate study populations to interrogate HDL function, protein modification, particle size, and protein cargo— each of which provides distinct information about HDL and its potential roles in biological processes. We observed altered HDL function, particle subpopulations, and protein cargo in smokers. Notably, HDL levels of alpha-1 antitrypsin, an acute phase protein, were positively associated with smoking status. These observations have implications for a role of smoking in generating modified forms of HDL that promote atherosclerosis. Table of Contents Chapter 1: Introduction ................................................................................................................1 Cholesterol Homeostasis Under Physiological Conditions..........................................................1 Figure 1.1 ............................................................................................................................2 Figure 1.2 ............................................................................................................................3 High Density Lipoprotein.............................................................................................................5 Figure 1.3 ............................................................................................................................6 Myeloperoxidase: A Mechanism For Loss of HDL Function......................................................9 Figure 1.4 ..........................................................................................................................12 Smoking .....................................................................................................................................13 Protease-Antiprotease Imbalance...............................................................................................15 Alveoli and Pulmonary Surfactant .............................................................................................17 Cigarette Smoking as a Possible Mechanism For Generating Dysfunctional HDL ..................21 Figure 1.5 ..........................................................................................................................22 Chapter 2: Materials and Methods ............................................................................................23 HDL Isolation.............................................................................................................................23 Trypsin Digest of HDL ..............................................................................................................24 Liquid Chromatography-Mass Spectrometry.............................................................................24 Tandem Mass Spectrometry Applied to Proteins.......................................................................27 Figure 2.1 ..........................................................................................................................28 Shotgun Proteomics ...................................................................................................................29 Targeted Proteomics...................................................................................................................30 Total Modified Tyrosine Analysis .............................................................................................30 PEG Precipitation of Plasma......................................................................................................31 Cholesterol Efflux Assays..........................................................................................................31 LCAT Activation Assay.............................................................................................................33 HDL/LDL Particle Analysis.......................................................................................................34 Protein Assay..............................................................................................................................35 SDS PAGE .................................................................................................................................35 Immunoblotting..........................................................................................................................35 CLEAR Subjects ........................................................................................................................36 Table 2.1 ...........................................................................................................................37 Smoking Cessation Subjects ......................................................................................................38 Statistical Analysis .....................................................................................................................38 Chapter 3: Discovery Analysis....................................................................................................40 Overview ....................................................................................................................................40 HDL Oxidation...........................................................................................................................40 Figure 3.1 ..........................................................................................................................41 Table 3.1 ...........................................................................................................................42 Figure 3.2 ..........................................................................................................................43 Figure 3.3 ..........................................................................................................................44 HDL Function ............................................................................................................................44 Figure 3.4 ..........................................................................................................................45 Figure 3.5 ..........................................................................................................................47 HDL Particle Concentration.......................................................................................................47 Figure 3.6 ..........................................................................................................................48 HDL Proteomics.........................................................................................................................49 Figure 3.7 ..........................................................................................................................50 Figure 3.8 ..........................................................................................................................51 Figure 3.9 ..........................................................................................................................54 Figure 3.10 ........................................................................................................................56 Conclusions ................................................................................................................................56 Chapter 4: Validation Analysis ..................................................................................................58 Overview ....................................................................................................................................58 Cholesterol Efflux ......................................................................................................................59 Figure 4.1 ..........................................................................................................................60 Figure 4.2 ..........................................................................................................................60 Table 4.1 ...........................................................................................................................61 Figure 4.3 ..........................................................................................................................61 HDL Particle Concentration.......................................................................................................62 Figure 4.4 ..........................................................................................................................62