A Second Look or, Not to Mention the Occasional Capsizing of a Windsurfer

Of all of the epithelial ovarian cancers (EOC), clear cell adenocarcinoma (CCA) has the worst clinical prognosis. Furthermore, the conventional EOC biomarker CA125 is more often negative in CCA than in other subtypes of EOC. This study sought to discover a new diagnostic biomarker that would allow more reliable detection of CCA. Using mass spectrometry, we compared proteins in conditioned media from cell lines derived from CCA and other types of EOC. We identified 30 extracellular or released proteins specifically present in CCA-derived cell lines. Bioinformatics analyses identified a serine protease inhibitor, tissue factor pathway inhibitor 2 (TFPI2), as a potential biomarker for CCA. Real time RT-PCR and Western blot analyses revealed that TFPI2 was exclusively expressed in CCA-derived cell lines and tissues. For clinical validation, we measured levels of TFPI2 and CA125 in a set of sera from 30 healthy women, 30 patients with endometriosis, and 50 patients with CCA, using an automated enzyme-linked immunosorbent assay systems. Serum levels of TFPI2 were significantly elevated in CCA patients, even those with normal CA125 levels. In terms of area under the receiver operating characteristic curve (AUC), TFPI2 was superior to CA125 in discriminating CCA patients from healthy women (AUC 0.97 for TFPI2 versus AUC 0.80 for CA125), or from patients with endometriosis (AUC 0.93 for TFPI2 versus 0.80 for CA125). This is the first evidence for TFPI2 as a serum biomarker of CCA. We propose that this biomarker may be useful for detection of CCA and for monitoring the transformation from endometriosis into CCA

Mass Spectrometric Analysis of the Phosphorylation Levels of the SWI/SNF Chromatin Remodeling/Tumor Suppressor Proteins ARID1A and Brg1 in Ovarian Clear Cell Adenocarcinoma Cell Lines

Protein phosphorylation is one of the major factors involved in tumor progression and malignancy. We performed exploratory studies aimed at identifying phosphoproteins characteristic to cell lines derived from ovarian clear cell adenocarcinoma (CCA), a highly malignant type of ovarian cancer. Comparative phosphoproteome analysis revealed that the phosphopeptides of five SWI/SNF chromatin remodeling/tumor suppressor components, including ARID1A and BRG1, were significantly down-regulated in CCA cells. We then quantitatively determined the phosphorylation levels of ARID1A and BRG1 by immunoprecipitation–multiple reaction monitoring (IP–MRM) that we used for analysis of the cognate phospho- and nonphosphopeptides of low-abundance proteins. The phosphorylation level of Brg1 at Ser1452 was down-regulated in CCA cells, whereas the phosphorylation level of ARID1A at Ser696 did not significantly differ between CCA and non-CCA cells. These results were consistent with the results of immunoblotting showing that Brg1 levels were comparable, but ARID1A levels were lower, in CCA cells relative to non-CCA cells. This is the first report to demonstrate reduced phosphorylation of Brg1 in CCA-derived cells. Our data also indicated that the IP–MRM/MS method we used is a powerful tool for validation of the phosphoproteins detected by shotgun analysis of phosphopeptides

N‑Myristoylation of the Rpt2 Subunit Regulates Intracellular Localization of the Yeast 26S Proteasome

The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of <i>Saccharomyces cerevisiae</i> and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome

N‑Myristoylation of the Rpt2 Subunit Regulates Intracellular Localization of the Yeast 26S Proteasome

The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of <i>Saccharomyces cerevisiae</i> and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome

N‑Myristoylation of the Rpt2 Subunit Regulates Intracellular Localization of the Yeast 26S Proteasome

The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of <i>Saccharomyces cerevisiae</i> and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome

Mass Spectrometric Identification of Glycosylphosphatidylinositol-Anchored Peptides

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs

Mass Spectrometric Identification of Glycosylphosphatidylinositol-Anchored Peptides

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs

Role of LRP1 and ERK and cAMP Signaling Pathways in Lactoferrin-Induced Lipolysis in Mature Rat Adipocytes

<div><p>Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and <i>in vitro</i> studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1.</p></div

Analysis of the effects of LF on the phosphorylation of HSL and PLIN by PKA and determination of PKA activity.

<p>Phosphorylation of HSL and PLIN by PKA was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to protein expression levels of HSL and PLIN are shown. <b>(A)</b> Phosphorylation of HSL Ser660 and <b>(B)</b> PLIN Ser497 by PKA. <b>(C)</b> Analysis of PKA activity in adipocytes treated with LF. PKA activity in adipocytes was detected using an ELISA before (0 min) and after treatment with LF. Kinase activity normalized to the total protein determined by BCA is shown. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *<i>p</i> < 0.05, ***<i>p</i> < 0.001 HSL, hormone-sensitive lipase; LF, lactoferrin; PLIN, perilipin; PKA, protein kinase A; SD, standard deviation.</p

LF-induced lipolysis in LRP1-silenced adipocytes.

<p><b>(A)</b> Activation of lipolysis by LF. To quantitate lipolysis, the amount of glycerol in the medium was analyzed 24 h after adding 1 mg/ml of LF. The statistical significance of the differences between LF treated and untreated cells was evaluated using the Student <i>t</i> test. **<i>p</i> < 0.01. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(B)</b> Activation of HSL by LF treatment. Phosphorylation of HSL was detected in the presence or absence of 1 mg/ml LF 15 min after the addition of LF. Phosphorylation levels normalized to protein expression levels are shown. The statistical significance was evaluated using the Student <i>t</i> test vs LF untreated control. **<i>p</i> < 0.01; n.s., no significant difference. The data represent the mean ± SD values of triplicate determinations of one of three identical experiments. <b>(C)</b> LRP1 silencing by siRNA. Adipocytes were transiently transfected with negative control siRNA (siNC) or LRP1 siRNA (siLRP1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141378#sec002" target="_blank">Materials and methods</a>). LRP1 protein expression was monitored by immunoblotting during each assay. Distinctive data is shown. β-actin was used as a loading control. HSL, hormone-sensitive lipase; LF, lactoferrin; LRP1, lipoprotein receptor-related protein 1; SD, standard deviation.</p