Dataset Information

Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells.

ABSTRACT: Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is important during placentation. Dysregulation of the process leads to pregnancy complications. S-nitrosylation of proteins is associated with cell invasion in many cell types. Adrenomedullin (ADM), a polypeptide expressed abundantly in the first-trimester placentas, induces EVCT invasion by upregulation of protein S-nitrosylation. This study aimed to identify the S-nitrosylated proteins induced by ADM in the JEG-3 placental cells. By using affinity chromatography followed by mass spectrometric analysis, tubulin, enolase, eukaryotic translation initiation factor 4A1, actin, annexin II (ANX II), and glyceraldehyde 3-phosphate dehydrogenaseprotein-1 were found to be S-nitrosylated by ADM. In vitro treatment with ADM or S-Nitrosoglutathione (GSNO) significantly increased the ANX II surface expression, but not its total expression in the JEG-3 cells. Translocation of ANX II to cell surface has been reported to act as a cell surface receptor to plasmin, plasminogen, and tissue plasminogen activator (tPA), thereby stimulating cell invasion and migration. However, in this study, ADM-induced surface expression of ANX II in the JEG-3 cells was not associated with changes in the secretory and membrane-bound tPA activities. Future studies are required to understand the roles of surface expression of S-nitrosylated ANX II on trophoblast functions. To conclude, this study provided evidences that ADM regulated the nitric oxide signaling pathway and modulated trophoblast invasion.

SUBMITTER: Li Y

PROVIDER: S-EPMC8907116 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells.

Li Yingting Y Zhong Liuying L Lee Cheuk-Lun CL Chiu Philip C N PCN Chen Min M

Reproductive sciences (Thousand Oaks, Calif.) 20210830 4

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is important during placentation. Dysregulation of the process leads to pregnancy complications. S-nitrosylation of proteins is associated with cell invasion in many cell types. Adrenomedullin (ADM), a polypeptide expressed abundantly in the first-trimester placentas, induces EVCT invasion by upregulation of protein S-nitrosylation. This study aimed to identify the S-nitrosylated proteins induced by ADM in the JEG ...[more]

PMID: 34462873

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Project description:Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 μM S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.

Project description: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.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 beta4, alpha-catenin, karyopherin [importin] beta1, 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.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.

Dataset Information

Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells.

Publications

Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells.

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