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Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells
BACKGROUND: Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pa...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816712/ https://www.ncbi.nlm.nih.gov/pubmed/20140087 http://dx.doi.org/10.1371/journal.pone.0009075 |
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author | Lam, Ying Wai Yuan, Yong Isaac, Jared Babu, C. V. Suresh Meller, Jarek Ho, Shuk-Mei |
author_facet | Lam, Ying Wai Yuan, Yong Isaac, Jared Babu, C. V. Suresh Meller, Jarek Ho, Shuk-Mei |
author_sort | Lam, Ying Wai |
collection | PubMed |
description | BACKGROUND: Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC. METHODOLOGY/PRINCIPAL FINDINGS: We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin β4, α-catenin, karyopherin [importin] β1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets. CONCLUSIONS/SIGNIFICANCE: We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells. |
format | Text |
id | pubmed-2816712 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-28167122010-02-07 Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells Lam, Ying Wai Yuan, Yong Isaac, Jared Babu, C. V. Suresh Meller, Jarek Ho, Shuk-Mei PLoS One Research Article BACKGROUND: Although overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC. METHODOLOGY/PRINCIPAL FINDINGS: We treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin β4, α-catenin, karyopherin [importin] β1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets. CONCLUSIONS/SIGNIFICANCE: We here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells. Public Library of Science 2010-02-05 /pmc/articles/PMC2816712/ /pubmed/20140087 http://dx.doi.org/10.1371/journal.pone.0009075 Text en Lam et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Lam, Ying Wai Yuan, Yong Isaac, Jared Babu, C. V. Suresh Meller, Jarek Ho, Shuk-Mei Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title | Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title_full | Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title_fullStr | Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title_full_unstemmed | Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title_short | Comprehensive Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial Cells |
title_sort | comprehensive identification and modified-site mapping of s-nitrosylated targets in prostate epithelial cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816712/ https://www.ncbi.nlm.nih.gov/pubmed/20140087 http://dx.doi.org/10.1371/journal.pone.0009075 |
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