Doctor of Philosophy, Case Western Reserve University, 2022, Molecular Medicine

Cardiovascular disease (CVD) is the leading cause of death worldwide. Traditional risk factors of CVD fail to elucidate the significantly increased CVD risk observed in patients with renal disease. Non-traditional risk factors are thought to contribute to this unexplained risk, through the action of lipoproteins. Historically the lipoprotein high- density lipoprotein (HDL) has been found to be cardio protective through reverse cholesterol transport in epidemiological studies. HDL is a heterogeneous particle comprised of a variety of constituents, the major constituent being apolipoproteinA-I (apoA-I). Post-translational modifications, including but not limited to carbamylation, chlorination, nitration and glycosylation, will render HDL dysfunctional. Evidence suggests that through a process known as protein carbamylation of apoA-I, the particle no longer functions in a cardio protective role but becomes pro-atherogenic. Protein carbamylation occurs through two different pathways. The first is a chemical reaction, which results in response to substantially increased levels of urea observed in chronic and ESRD. The second enzymatic reaction occurs during leukocyte activation of myeloperoxidase (MPO, at sites of inflammation. This process results in the addition of a “carbamoyl” moiety to amines of proteins and amino acids. Studies have been performed analyzing (carbamylated HDL) c-HDL function but have not identified the relationship of site-specific carbamylation of apoA-I/ HDL within human atheroma. As declining kidney function potentially alters the structure and function of c-HDL, the effect of carbamylation becomes increasingly relevant in understanding the pathophysiology of CVD progression. Our goal is to expand our understanding of HDL structurally and clinically through uncovering the site-specific protein carbamylation patterns. We hypothesized that insights into the chemical environment within the human artery wall could be gained by monitoring site-specif (open full item for complete abstract)

Committee: Stanley Hazen (Advisor) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2020, Molecular Medicine

Objective: Prostate cancer is the second leading cause of cancer-related deaths among men in the US. Although some reports show high concentrations of HDL cholesterol increase risk for prostate cancer, this association has not been consistent. High density lipoprotein (HDL) metabolism, is facilitated largely by scavenger receptor class B, type 1 (SR-B1) that mediates its uptake into cells, and ABCA1 that mediates its generation. SR-B1 is upregulated in prostate cancer tissue, whereas some evidence suggests that ABCA1 is downregulated in the disease. Our efforts were to determine if SR-B1-dependent HDL uptake and/or HDL biogenesis by ABCA1 export of lipids to apoA1 promotes prostate cancer cell proliferation and disease progression. Hypothesis: HDL uptake by SR-B1 drives prostate cancer proliferation and disease progression, whereas ABCA1 mediated lipid efflux decreases prostate cancer proliferation and disease progression (Fig. Abstract) Methods and Results: Here, we report that knockout (KO) of SR-B1 via CRISPR/Cas9 editing led to reduced HDL uptake into prostate cancer cells, and reduced their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 wildtype (SRB1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice showed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts, than SR-B1+/+ cells in same host model. Tumor volume data was overall consistent survival data. Conclusion: The results suggest that HDL through tumoral SR-B1 significantly influences the proliferation of prostate cancer cells and is a driver of the disease. Further investigation is needed to conclusively determine how HDL metabolism by ABCA1 influences prostate cancer cells and its impact on disease progression.

Committee: Jonathan Smith PhD (Advisor); Angela Ting PhD (Committee Chair); W.H. Wilson Tang MD (Committee Member); J. Mark Brown PhD (Committee Member); Nima Sharifi MD (Committee Member) Subjects: Cellular Biology; Medicine; Molecular Biology; Oncology

PhD, University of Cincinnati, 2018, Medicine: Pathobiology and Molecular Medicine

Background: Atherosclerosis is a multifactorial inflammatory disease that begins with the accumulation of lipid in arterial endothelium. Atherosclerosis is the leading cause of deaths attributable to cardiovascular disease in the United States. Epidemiological studies showing high-density lipoprotein (HDL) cholesterol is inversely correlated with atherosclerosis has made it a pharmacological target for preventing cardiovascular disease. However, outcomes from clinical trials have raised questions about HDL's protective properties. Investigating the molecular interactions of apolipoprotein (apo)A-I, which accounts for approximately 70% of total HDL protein, can help translate HDL structure to cardioprotective function. Lecithin: cholesterol acyl transferase (LCAT) is a critical HDL-modifying protein that performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring-structure that encapsulates lipid in reconstituted (r)HDL. Cross-linking analysis of rHDL implies that apoA-I molecules exist in at least two distinct organizations: one with helix 5 of an apoA-I molecule adjacent to helix 5 of its antiparallel partner (5/5 helical registry), and the other in a 5/2 registry. We hypothesized that the orientation of apoA-I molecules on rHDL modulates LCAT activity. Objective: Identify the mechanism by which apoA-I activates LCAT to determine how HDL interacts with its immense proteome. Linking HDL structure and function will allow for therapeutic development that targets HDL-associated inflammatory diseases. Major Findings: 1) Antiparallel apoA-I molecules adopt a thumbwheel mechanism to generate a discontinuous epitope for LCAT activation. Site-directed cysteine mutagenesis was used to “lock” two apoA-I molecules into an antiparallel 5/5, 5/2, and 5/1 helical registry on rHDL. The 5/5 mutant demonstrated higher LCAT acti (open full item for complete abstract)

Committee: William Sean Davidson Ph.D. (Committee Chair); Christopher A. Crutchfield Ph.D. (Committee Member); Philip Howles Ph.D. (Committee Member); Francis McCormack M.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); Laura Woollett Ph.D. (Committee Member) Subjects: Pathology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2015, College of Sciences and Health Professions

It is generally accepted that apolipoprotein A-I (apoA-I), the main protein constituent of high density lipoprotein (HDL), forms a dimeric antiparallel structure that confers nascent HDL function and stability and provides a scaffold for the lipid phase. Moreover, while the truncated lipid-free apoA-I crystal structures suggest more compact helical bundle structure, the solution structure of full length lipid-free apoA-I is unclear, and the mechanism of how monomeric lipid-free apoA-I associates in antiparallel fashion during HDL formation is shrouded in mystery. Here in this study we performed hydrogen-deuterium exchange (HDX) and lipidation kinetic analyses of monomeric and multimeric (predominantly dimeric) apoA-I employing both mass spectrometry (HDX-MS) and functional lipidation assays. At lower concentration (0.07 mg/ml) lipid-free apoA-I is monomeric (based on equilibrium PAGE and cross-linking studies) and exhibits multiple discrete yet reproducible regions with bimodal HDX kinetics indicative of the existence of distinct populations of molecules that have domains with alternative secondary structure (random coil vs. helical). Kinetic HDX studies further reveal that at higher concentration (0.7 mg/ml) lipid-free apoA-I self-association promotes a conformational change of the C-terminus domain E223-A232 from random coil to alpha-helix. Surprisingly, apoA-I self-association seems to bury E223-A232 within the binding interface, which significantly slows down the rate of apoA-I lipidation. Peptide competition assays show that N- and C-terminus peptides (K12-D24 and E223-A232, respectively) interfere with apoA-I self-association, but only the C-terminus peptide blocks apoA-I lipidation. Taken together, the findings of this thesis indicate that HDL genesis starts with lipidation of apoA-I monomers followed by apoA-I dimerization in an antiparallel fashion on the lipid surface.

Committee: Valentin Gogonea PhD (Committee Chair); Stanley Hazen MD. PhD (Committee Member); Jonathan Smith PhD (Committee Member); David Anderson PhD (Committee Member); Joseph Didonato PhD (Committee Member); Mekki Bayachou PhD (Committee Member) Subjects: Biochemistry

MA, Kent State University, 2014, College of Arts and Sciences / School of Biomedical Sciences

Wrood Salim Al-Khfajy, M.A., December 2014 Pharmacology Role of Transmembrane 141 in Cholesterol Metabolism Thesis Advisor: Yanqiao Zhang TMEM141 belongs to the large family of transmembrane (TMEM) proteins, and its physiological role remains largely unknown. In our study, we found that hepatic Tmem141 expression is markedly induced by FXR and repressed in db/db or high fat diet-fed mice. We determined the effect of acute loss or augmentation of hepatic Tmem141 function on cholesterol homeostasis. We generated adenovirus expressing LacZ (Ad-shLacZ) or small hairpin RNA of Tmem141 (Ad-shTmem141). Hepatic knockdown of Tmem141 markedly reduced hepatic Tmem141 expression and resulted in striking phenotypes, including a >8-fold decrease in total plasma cholesterol and a >80% decrease in HDL-C and LDL-C. Consistent with the loss-of-function data, Tmem141 deficiency reduces ABCA1 expression in human hepatic (Huh-7) cells, and macrophages and mouse hepatocytes, and causes impaired cholesterol efflux in both macrophages and hepatocytes. Interestingly, mice overexpressing hepatic Tmem141 had unchanged plasma cholesterol levels and ABCA1 expression. Co- immunoprecipitation assays demonstrate a direct interaction between Tmem141 and ABCA1. Finally, in vitro study revealed that Tmem141 is ubiquitously expressed and localizes mainly to endocytic compartments where it may regulate ABCA1 degradation/recycling. In summary, we have identified Tmem141 as a novel posttranscriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, a better understanding of the pathways, cells use to initiate HDL assembly is necessary to design novel therapies to increase HDL formation clinically to reduce cellular cholesterol pool by promoting reverse cholesterol transport from cells and preventing atherosclerotic vascular disease.

Committee: Yanqiao Zhang M.D. (Advisor); Yoonkwang Lee Ph.D. (Committee Member); Werner Geldenhuys Ph.D. (Committee Member) Subjects: Pharmacology

PhD, University of Cincinnati, 2012, Engineering and Applied Science: Biomedical Engineering

High density lipoproteins are a heterogeneous group of particles composed of proteins and lipids that in approximately equal mass. The most abundant HDL proteins are apolipoprotein (apo) A-I and A-II, yet recent proteomic studies have identified up to 50 additional proteins within HDL. HDL is well-known for its critical cardiovascular disease (CVD) prevention function, which is mainly achieved through the mediation of the reverse cholesterol transport (RCT). In addition to that, recent studies have shown that HDL also displays a series of similarly diverse functions related with CVD-protection, including anti-oxidation, anti-inflammation, and endothelial relation. What's more, a growing body of evidence (including our research) has suggested these diverse HDL functions may be mediated by distinct stable subspecies that happen to co-fractionate with classically defined “HDL”. To better characterize the structural composition and functionality of HDL subspecies, our collaborator have applied three non-density based orthogonal separation chromatography techniques (Gel filtration (GF), Anion exchange (AE), and Isoelectric focusing (IEF)) for the isolation of HDL from human plasmas. Generally, these techniques fractionated normal human plasmas to phospholipid-containing subfractions, then the HDL associated proteins and their distributions were determined using Mass Spectrometry. Given the proteomic profiles of HDL proteins, our work is to systematically identify the structural HDL subspecies and study their biological functions. In the first step, we assume that HDL associated proteins, which have similar co-migration patterns when separated by different techniques, are likely to form distinct lipoprotein subspecies. So for a protein pair, showing consistently high similarity in migration patterns across techniques would provide the strongest evidence of their co-existence in the same particle. Therefore, we developed two novel scoring systems to quantitatively measure (open full item for complete abstract)

Committee: Long Lu PhD (Committee Chair); Sean Davidson PhD (Committee Member); Jaroslaw Meller PhD (Committee Member); Marepalli Rao PhD (Committee Member) Subjects: Bioinformatics

PhD, University of Cincinnati, 2010, Medicine: Pathobiology and Molecular Medicine

High density lipoprotein (HDL) plasma levels are inversely correlated with the risk of developing cardiovascular disease. HDL is formed when lipid-free apolipoproteins in the plasma accept excess phospholipids and cholesterol from cells such as hepatocytes, enterocytes, and macrophages. This process is mediated by a cell membrane protein known as ATP-binding cassette transporter A1 (ABCA1). It is unknown what structural elements in apolipoproteins allow them to participate in ABCA1-mediated cholesterol efflux. The hypothesis tested in this work is that amphipathic and charged helical structural elements of exchangeable apolipoproteins allow these proteins to facilitate ABCA1-mediated cholesterol efflux at the cell surface. Recently, it was proposed that a negatively charged and a hydrophobic surface patch on apolipoprotein (apo) A-I were important in this process (1). Our data shows that neither of these surface patches plays an important functional role in apoA-I promoted cholesterol efflux via ABCA1. It has also been proposed that a linear array of acidic amino acids aligned along the junction of the hydrophobic and hydrophilic faces of two amphipathic α-helices is the critical element for this process (2). However, studies using apoC-I point mutants demonstrated that this element was also functionally unnecessary. Instead, our studies with peptides modeling the amphipathic α-helices of apoA-II and apoC-I have shown that the minimal structural unit in apolipoproteins which allows them to serve as cholesterol acceptors in ABCA1-medated efflux is a bihelical peptide composed of an amphipathic non-lipid binding helix joined to an amphipathic fast lipid binding helix. In apoA-I, apoA-II, apoC-I, and likely apoE this structural element is found at the extreme C-terminus of the protein with the fast lipid binding helix being closest to the C-terminus. It was found that the non-lipid binding helix altered the phospholipid binding preference of the fast lipid binding heli (open full item for complete abstract)

Committee: Sean Davidson PhD (Committee Chair); Laura Conforti PhD (Committee Member); Thomas Thompson PhD (Committee Member); David Hui PhD (Committee Member); Lois Arend PhD, MD (Committee Member); Melanie Cushion PhD (Committee Member) Subjects: Molecular Biology

PhD, University of Cincinnati, 2004, Medicine : Pathobiology and Molecular Medicine

Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity and mortality in the United States due to consumption of a high-fat “Western” diet. A major indication of these disease states is excess lipid deposition, particularly cholesterol, in the arterial walls. It has been well documented that high levels of circulating high-density lipoprotein (HDL) and its main protein component apolipoprotein AI (apoA-I) are associated with lowered risk of cardiovascular disease. The protective effects of HDL are thought to be mediated by a process called reverse cholesterol transport - in which HDL takes up excess cholesterol from peripheral tissues and transports it to the liver for excretion in the bile. It is now widely accepted that the interaction of apoA-I with the cell membrane protein ATP-binding cassette transporter AI (ABCAI) is critical for the formation of nascent HDL particles. Since sphingomyelin maintains a preferential interaction with cholesterol in membranes, the breakdown of sphingomyelin may regulate the availability of cellular cholesterol utilized by ABCAI. Furthermore, the catabolite of sphingomyelin, ceramide, is a potent signaling molecule and may play an important role in ABCAI regulation or function. The following study examines the potential contribution of sphingolipids in ABCAI-mediated cholesterol removal from the cell. It was discovered that treatment with C2-ceramide enhances cholesterol release to apoA-I. This effect appeared to becaused by an increase in cellular ABCAI content with enrichment at the cell surface. These findings may lead to new ways to increase cellular ABCAI and further promote cholesterol removal from regions of excess cholesterol such as the atherosclerotic lesion.

Committee: Dr. William Davidson (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2010, Cell Biology

High levels of high density lipoprotein (HDL) are associated with a decreased risk of cardiovascular disease (CVD). The atheroprotective function of HDL has been attributed to its key role in the reverse cholesterol transport (RCT) pathway. However, recent evidence suggests that HDL can be rendered “dysfunctional”, impairing its ability to promote RCT. The work described here suggests two mechanisms that can render HDL “dysfunctional.” The first mechanism involves oxidation of HDL by the enzyme myeloperoxidase (MPO). Recent studies demonstrate that MPO binds to HDL in vivo, selectively targeting HDL for oxidative modification. We now show that (patho) physiologically relevant levels of MPO-catalyzed oxidation result in loss of non cholesterol efflux activities of HDL including anti-apoptotic and anti-inflammatory functions. One mechanism responsible is shown to involve loss of oxidized HDL binding to the HDL receptor, scavenger receptor B1, and concurrent acquisition of binding to a novel unknown receptor independent of scavenger receptors CD36 and SR-A1. HDL modification by MPO is further shown to confer pro-inflammatory gain of function activities as monitored by NF-kappa B activation and surface vascular cell adhesion molecule (VCAM-1) levels on aortic endothelial cells. Multiple site-directed mutagenesis studies of HDL suggest that the pro-inflammatory activity does not involve methionine, tyrosine, or tryptophan residues—oxidant sensitive residues previously mapped as sites of oxidation within human atheroma. A second mechanism for generating dysfunctional HDL involves changing the apolipoprotein composition. Apolipoprotein A2 (apoA2) is the second most abundant protein in HDL. However, the role of apoA2 in the atheroprotective function of HDL is not well defined. We now show that apoA2 containing HDL is less anti-apoptotic and less anti-inflammatory than HDL containing only apoA1. Further, oxidation of apoA2 containing HDL by MPO generates a particle that has (open full item for complete abstract)

Committee: Stanley Hazen (Advisor); Alan Levine (Committee Chair); Jonathan Smith (Committee Member); Menachem Shoham (Committee Member); Mark Chance (Committee Member) Subjects: Cellular Biology

PhD, University of Cincinnati, 2012, Medicine: Pathobiology and Molecular Medicine

High density lipoproteins (HDL) are complexes of phospholipid, cholesterol and protein that circulate in the blood. Epidemiological studies have demonstrated a strong inverse correlation between plasma levels of HDL associated cholesterol (HDL-C) and the incidence of cardiovascular disease (CVD). Clinically, HDL-C is often measured and used in combination with low density lipoprotein cholesterol (LDL-C) to assess overall cardiovascular health. HDL have been shown to possess a wide variety of functional attributes which likely contribute to this protection including anti-inflammatory and anti-oxidative properties and the ability to remove excess cholesterol from peripheral tissues and deliver it to the liver for excretion, a process known as reverse cholesterol transport. This functional diversity might be explained by the complexity of HDL composition. Recent studies have taken advantage of advances in mass spectrometry technologies to characterize the proteome of total HDL finding that over 50 different proteins can associate with these particles. This adds to a growing body of evidence that supports the global hypothesis of this thesis which is that the total pool of HDL in an individual is composed of numerous subspecies with distinct protein and lipid compositions and therefore will have distinct functional properties. Additionally, we believe that the composition of HDL is dynamic and can change in response to changes in the environment of the blood, as can occur in disease. To test these hypotheses we devised an approach based on three aims. Aim 1: Identify and characterize HDL subspecies based on protein composition. Aim 2: Analyze functional heterogeneity across separated plasma HDL fractions. Aim 3: Examine the effect of type 2 diabetes on HDL subspecies distribution in young adults. To accomplish these goals we have developed novel methods for the separation and fractionation of HDL subspecies from human plasma and their subsequent proteomic and functional (open full item for complete abstract)

Committee: Sean Davidson Ph.D. (Committee Chair); David Askew Ph.D. (Committee Member); Long Lu Ph.D. (Committee Member); Francis McCormack M.D. (Committee Member); Ranasinghe Silva Ph.D. (Committee Member) Subjects: Surgery