A DISCOVERY-DRIVEN LABEL-FREE PROTEOMICS BASED SURVEY OF ESTRADIOL-REGULATED PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL FUNCTION IN THE RAT RETINA

Abstract

A DISCOVERY-DRIVEN LABEL-FREE PROTEOMICS BASED SURVEY OF ESTRADIOL-REGULATED PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL FUNCTION IN THE RAT RETINA Khadiza Zaman1, Fatima Rahlouni1,2, Vien Nguyen1, Katalin Prokai-Tatrai1 and Laszlo Prokai1 1 Department of Pharmacology & Neuroscience,University of North Texas Health Science Center, Fort Worth, TX. 2 Texas College of Osteopathic Medicine, University of North Texas Health Science Center, Fort Worth, TX. Purpose: Previous studies have established the pleiotropic role of 17β-estradiol (the predominant human estrogen) as a potent neuroprotectant, but only recently it has gained attention for its therapeutic potential against ocular neurodegenerative diseases. Thus, this study was designed to perform a label free quantitative proteomics based survey to understand the impact of topical administration of E2 on the rat retina. This is one of the first reports elucidating E2-regulation of rat retinal proteins, networks and associated biological processes, thus providing us with more insights on topical hormone therapy. Methods: Ovariectomized (OVX) Brown Norway rats were given 0.1% w/v E2 eye drops in saline/2-hydroxypropyl-β-cyclodextrin vehicle and controls received vehicle daily for three weeks. Retina from euthanized animals were immediately isolated. Retinal proteins were extracted and analyzed using data-dependent nanoflow LC-ESI-MS/MS on Orbitrap EliteTM (Thermo) or Orbitrap Velos Pro. MS/MS data was searched against the UniProt rat protein database using Mascot (Matrix Science). Validations and label-free quantitation were performed using Scaffold (Proteome Software) by observing changes in protein abundances between treated and control using t test. Differentially expressed proteins were mapped to protein interaction networks and biological processes through Ingenuity Pathway Analysis® (Qiagen). Results: In our proteomics-based quantitation, we identified 66 E2 regulated proteins in the OVX rat retina among which 49 up-regulated and 17 down-regulated (p1.5-fold change between groups). Some of the most highly scored identified networks are associated with endocrine system disorders, organismal injury and abnormalities, and developmental disorder. Presence of nuclear estrogen receptor (ER) in our dataset also reinforces the intricate nature of E2 signaling conveying neuroprotection. Our network-based analysis emphasized on the role of E2 in neuroprotection through regulation of various stress-induced signaling cascades such as ERK/MAPK pathways. Conclusion: By using an OVX model with little or no endogenous E2, our study potentiates the neuroprotective role of E2 upon topical administration of the hormone. With this vast array of information on estrogen biology we seek to create foundations in basic science research regarding hormone therapy focusing on the “estrogenic retina.” Acknowledgement: This study was supported by the National Eye Institute and the Office of Research on Women’s Health (grant number EY027005 to K.P.-T.) and by the Robert A. Welch Foundation (endowment BK-0031 to L.P.).