Molecular Response of Retinal Pigment Epithelial Cells to Oxidized Lipoproteins: Global and Targeted Studies
Global-scale examinations of biological systems at the molecular level complement targeted approaches to scientific inquiry that focus on specific subsets of biomolecules, or on a single molecule of interest. In this dissertation, we utilized both the discovery-based approach to evaluate the proteomics workflows centered around mass spectrometry as the key technology, and the targeted approach to examine the molecular response of RPE due to oxidized lipoproteins (oxLDL) treatments. A crucial aspect in proteomics studies is the design of bioanalytical strategies that maximize coverage of the complex repertoire of a proteome. A comprehensive, unbiased examination of the proteome represents a powerful approach toward system-level insights into disease mechanisms. We evaluated the performance of bioanalytical platforms for profiling of the proteome in a biological system. We applied a discovery-based approach to evaluate the global transcriptome and proteome changes due to oxLDL treatment in ARPE-19 cells. We studied the role of scavenger receptors CD36 and CD5L/AIM in ARPE-19 cells when induced with oxLDL. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations. We applied a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in a data-dependent setting and used bioinformatics for protein identification. The three platforms identified a total of 1043 proteins altogether. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage. We also evaluated another bioanalytical platform which consists of a highresolution mass spectrometer combined with nano-UPLC in a data-independent setting without pre-fractionation for oxLDL mediated proteome alteration in ARPE-19 cells. This platform outperformed the SDS-PAGE based analytical platform in terms of proteome coverage as it identified around 2500 proteins, ca. 3-fold more proteins than the latter. Most importantly, this platform was able to perform label free quantification of differentially expressed proteome alteration. The platforms identified proteins with diverse physicochemical characteristics involved in various functional roles within the biological system. Furthermore, we carried out the first comparative transcriptomic and proteomic study for the evaluation of oxLDL effects on ARPE-19 cells after a 4 h exposure. The treatment with oxLDL affected the regulation of more than 700 genes that were involved in regulation of cell cycle, oxidative stress, cholesterol efflux, circadian rhythm, NRF-2 pathways. However, LDL treatment alone did not induce the regulation of these pathways. The differential proteomic analysis found 41 proteins affected due to the oxLDL treatment. This study provided a foundation for a bioanalytical platform for identification and label-free quantification in the human retinal pigment epithelial cells (ARPE-19) proteome. The list of differentially expressed proteins due to oxLDL treatment identified in this study gives insights to the change in proteins that might be interrogated for their roles in pathogenesis of macular degeneration. These findings could give us targets to intervene in the pathogenesis of AMD progression in human for the development of better treatment and prevention against this degenerative disease. Lastly, we studied the mechanistic role of scavenger receptors CD36 and CD5L/AIM in oxLDL uptake by ARPE-19 cells. We, for the first time, demonstrated the presence of scavenger receptor CD5L in ARPE-19 cell. The oxLDL uptake was primarily dependent on CD36, and both the CD5L/AIM and CD36 were seen to co-localize in the presence of oxLDL. Our results suggest a new dynamics on CD5L/AIM on the oxLDL uptake that was not seen in macrophages. The reduction in intracellular accumulation of oxLDL in the presence of extracellular recombinant CD5L/AIM is an interesting phenomenon as it has been recently shown the involvement of CD5L/AIM in autophagy.
Age Related Macular Degeneration, Bioanalytics, Mass Spectrometry, Proteomics, Retinal Pigment Epithelium, Medicine and Health Sciences
University of Tennessee Health Science Center
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