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Nitric Oxide-Mediated Signaling in Pulmonary Endothelial Cells

Stitt-Fischer, Molly Sue (2008) Nitric Oxide-Mediated Signaling in Pulmonary Endothelial Cells. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

S-nitrosothiol modifications of proteins are emerging as an important nitric oxide-mediated signaling pathway. Our laboratory has focused on S-nitrosation of the metal binding protein metallothionein and the resulting effects on zinc homeostasis, gene and protein expression and nitric oxide (NO) mediated signaling in the pulmonary endothelium. Statement of public health significance: The pulmonary endothelium is responsible for filtering the blood before it enters systemic circulation, and as such it is extremely vulnerable to injury by inhaled toxicants in the environment as well as those that circulate in the bloodstream. As the endothelium constitutively produces NO, we are interested in studying NO-mediated signaling in order to lay a foundation that will allow us to better understand diseases such as asthma, pulmonary hypertension and sepsis in which dysregulation of NO-mediated signaling is thought to be a contributing factor to the disease pathology. To this end we have used both recombinant DNA and biochemical techniques to examine the relationship between metallothionein, zinc homeostasis and the metal responsive transcription factor MTF-1. We demonstrated that exposure to NO results in zinc release from metallothionein, which in turn activates MTF-1, resulting in nuclear translocation of the protein and NO-dependent increases in metallothionein protein expression. We hypothesized that S-nitrosation of the sulphydryl groups in metallothionein were the cause of NO-mediated zinc release and downstream protein expression effects. We used a fluorescent modification of the biotin switch assay in combination with two-dimensional electrophoresis and mass spectroscopy to extend our study of NO-mediated signaling through S-nitrosation of protein thiols to identify S-nitrosated metallothionein in endothelial cells exposed to NO donor, and used the technique in further studies to illuminate the proteome of pulmonary endothelial cells. We were able to identify several potential targets of S-nitrosation in endothelial cells including cytoskeletal, cytoprotective, glycolytic and chaperone proteins. The proteomic assay that we developed is a useful screening tool, and may lead to new insights in post-translational S-nitrosothiol modifications of endothelial proteins, and eventually to new perspectives regarding diseases exacerbated by dysregulation of this NO-mediated signaling pathway.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Stitt-Fischer, Molly Suemstittfischer@mac.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairPitt, Brucebrucep@pitt.eduBRUCEP
Committee MemberBarchowsky, Aaronaab20@pitt.eduAAB20
Committee MemberChoi, Augustineamchoi@rics.bwh.harvard.edu
Committee MemberSt Croix, Claudettecls13@pitt.eduCLS13
Date: 26 June 2008
Date Type: Completion
Defense Date: 2 April 2008
Approval Date: 26 June 2008
Submission Date: 11 April 2008
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Environmental and Occupational Health
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Biotin Switch; endothelial cells; endothelium; metallothionein; MTF-1; Nitric oxide; proteomics; S-nitrosation; zinc
Other ID: http://etd.library.pitt.edu/ETD/available/etd-04112008-105942/, etd-04112008-105942
Date Deposited: 10 Nov 2011 19:35
Last Modified: 15 Nov 2016 13:39
URI: http://d-scholarship.pitt.edu/id/eprint/7008

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