Abstract
BackgroundAlthough 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 FindingsWe 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/SignificanceWe 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.
Highlights
Evidence suggesting that an impaired nitric oxide (NO)signaling contributes to the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer (PCa) is accumulating
Immunohistologic studies demonstrated that iNOS is not expressed in normal prostate [8] but that the enzyme is expressed in all specimens with BPH, low- or high-grade prostatic intraepithelial neoplasia (PIN), and PCa. iNOS immunoreactivity was found to be higher in high-grade PIN and PCa than in BPH and low-grade PIN
nitrosative stress (NS) has been proposed to promote the development of prostate disease, partly by imparting damage to DNA, proteins, and lipids [3,9,10,11,12], we lack a clear understanding of the mode of action of NS, despite recent reports suggesting that the aggressiveness of PCa cell lines can partly be determined by NO [13,14]
Summary
Evidence suggesting that an impaired nitric oxide (NO)signaling contributes to the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer (PCa) is accumulating This view lends supports to the postulate that chronic prostatic inflammation is an inciting factor for BPH and PCa [1,2] or that proliferative inflammatory atrophy is the precursor of PCa [3]. Studies of this relationship remain sparse and the major findings are limited to a few reports of an aberrant expression of the inducible nitric oxide synthase (iNOS or NOS-2) in diseased prostate tissues [4,5,6,7]. Our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC
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