Browsing by Author "Rahlouni, Fatima"
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Item A DISCOVERY-DRIVEN LABEL-FREE PROTEOMICS BASED SURVEY OF ESTRADIOL-REGULATED PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL FUNCTION IN THE RAT RETINA(2019-03-05) Rahlouni, Fatima; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo; Zaman, KhadizaA 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.).Item Identification of Estrogen-Regulated Proteins in Zebrafish Embryos by Quantitative Proteomics(2016-03-23) Shulaev, Vladimir; Prokai, Laszlo; Rahlouni, FatimaShort description: In this presentation, we introduce quantitative proteomics using zebrafish embryos as a paradigm for testing the potential impact of endocrine disrupting chemicals that represent a class of exogenous compounds interfering with the biological actions of endogenous hormones. Specifically, we aimed at evaluating the effect of deyolking (which is a generally employed procedure to better identify low abundance proteins in embryos) on the results of label-free protein quantification, as well as understanding protein networks affected by estrogens by exposing this model organism to 17β-estradiol (E2). Purpose: To identify estrogen-induced differential protein expression impacted by deyolking in zebrafish embryos using a label-free quantitative proteomics approach. Methods: Along with non-treated controls, zebrafish embryos were treated short-term with 1 ppm of E2. Half of the embryos were subjected to a deyolking procedure. Embryo protein extracts were processed using a bottom-up shotgun proteomics approach. The samples were analyzed using data-dependent LC-ESI-MS/MS on an LTQ-Orbitrap Velos Pro (Thermo) connected to a nano-ACQUITY UPLC system (Waters). MS/MS spectra were searched against a composite UniProt zebrafish protein database using the Mascot and SEQUEST search algorithms within Proteome Discoverer (Thermo Scientific). Label-free quantitation was performed by an MS/MS total ion current approach using Scaffold (Proteome Software), as well as calculating spectral counts. Additionally, the differentially expressed proteins were mapped to networks and biological processes through Ingenuity Pathway Analysis (IPA, QIAGEN). Results: The increase in the zebrafish yolk protein vitellogenin is a well-known marker of estrogen exposure. With the observation of a significant increase in vitellogenin in the treated embryos compared to non-treated control embryos, we confirmed E2’s action for our subsequent proteomics study. Estrogen-regulated proteins were identified using both spectral counting and MS/MS-based total ion current method as label-free quantitative approaches. With p2-fold change as threshold, 74 proteins were differentially regulated by the hormone by combining data from both yolk-intact and deyolked samples. Of these significantly differentially regulated proteins, 3 were found to be unique to spectral counting, while 7 were unique to yolk-intact samples. Three proteins were represented in both yolk-intact and deyolked specimen but to differing degrees of up- and down-regulation. When the differentially regulated 74 proteins were submitted for pathway analysis, 53 proteins were mapped into 1 network that merged into an E2-regulated pathway. We saw repression of several proteins such as ATP synthase alpha- and beta-subunits and eukaryotic elongation factor 2 in E2-treated embryos. However, in other models, these proteins were established previously to be activated by estrogen. Therefore, the deyolking procedure significantly alters the state of the proteome in such a way that it potentially invalidates the results of quantitative proteomics studies. Conclusions: The deyolking of zebrafish embryos to increase protein coverage alters expression data obtained by quantitative proteomics. (Supported by the Robert A. Welch Foundation, BK-0031)Item Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications(MDPI, 2021-02-08) Prokai, Laszlo; Rahlouni, Fatima; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, KatalinThe widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17beta-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.Item Retina-Targeted Delivery of 17beta-Estradiol by the Topically Applied DHED Prodrug(MDPI, 2020-05-16) Prokai-Tatrai, Katalin; Nguyen, Vien; De La Cruz, Daniel L.; Guerra, Rebecca; Zaman, Khadiza; Rahlouni, Fatima; Prokai, LaszloThe purpose of this study was to explore retina-targeted delivery of 17beta-estradiol (E2), a powerful neuroprotectant, by its bioprecursor prodrug 10beta,17beta-dihydroxyestra-1,4-dien-3-one (DHED) administered as eye drops in animal models. Compared to the parent hormone, DHED displayed increased transcorneal flux ex vivo both with and without the presence of 2-hydroxypropyl-beta-cyclodextrin used as a penetration-enhancing excipient in rat, rabbit, and pig. In vitro, the prodrug also showed facile bioactivation to E2 in the retina but not in the cornea. After topical administration to rats and rabbits, peak DHED-derived E2 concentrations reached 13 +/- 5 ng/g and 18 +/- 7 ng/g in the retina of female rats and rabbits, respectively. However, the prodrug remained inert in the rest of the body and, therefore, did not cause increase in circulating hormone concentration, as well as wet uterine and anterior pituitary weights as typical markers of E2's endocrine impact. Altogether, our studies presented here have demonstrated the premise of topical retina-selective estrogen therapy by the DHED prodrug approach for the first time and provide compelling support for further investigation into the full potential of DHED for an efficacious and safe ocular neurotherapy.Item Transcorneal Permeability Pilot Studies(2018-03-14) Nguyen, Vien; Johnston, Joseph; Prokai-Tatrai, Katalin; Rahlouni, FatimaPurpose: Permeability of potential ocular agents across the cornea is important in evaluating their ability to enter the eye by the transcorneal route. Accordingly, the purpose of this study was to set up a transcorneal permeability protocol and develop the necessary analytical methods to evaluate flux differences among test agents across cornea from different species. Methods: Corneas from rat, pig and rabbit were used. Jacketed vertical diffusion cells were used to test transcorneal permeability. Two test compounds of comparable molecular volume but differing in lipophilicity (measured by the logarithm of the n-octanol/water partition coefficient, logP) by approximately two log units were chosen for this pilot study. Once cornea and cell chambers were equilibrated to 35°C, cornea was mounted and test compounds at a given concentration either in saline or in saline containing a permeability enhancer excipient were placed in the donor chamber. The amount of test agents in the receiver chamber versus time was measured based on methods developed in our laboratory and using LC-MS/MS selected reaction monitoring on a TSQ Quantum Ultra (Thermo) connected to a Surveyor HPLC system (Thermo). Quantification of each test compound was performed based on the principles of stable isotope dilution. Flux was calculated, based on the principles of diffusion testing. Results: We were able to establish the transcorneal permeability procedure for testing the diffusion of compounds across the cornea. In addition, selected reaction monitoring LC-MS/MS quantification methods were developed for the test compounds. Of our two test compounds in this pilot study, we were able to show that the flux across the cornea for test compound with intermediate lipophilicity was approximately one log unit higher for all species when saline vehicle was used and about 0.5 log unit higher when a permeability enhancer excipient was used, compared to those of test agent with high lipophilicity (logP about 4). Conclusions: This pilot study showed the establishment of a protocol for a convenient evaluation of the transcorneal permeability of test compounds quantified by LC-MS/MS analyses. This study was supported by NIH grant R01EY027005 (KPT)