Noriaki Arakawa

Co- and post-translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including Nα‐acetylation, Nα‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.

Secretome-based identification of TFPI2, a novel serum biomarker for detection of ovarian clear cell adenocarcinoma.

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.

The novel PAR-1-binding protein MTCL1 has crucial roles in organizing microtubules in polarizing epithelial cells.

Summary The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiled-coil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.

Wild-type p53 enhances annexin IV gene expression in ovarian clear cell adenocarcinoma

The protein annexin IV (ANX4) is elevated specifically and characteristically in ovarian clear cell adenocarcinoma (CCA), a highly malignant histological subtype of epithelial ovarian cancer. On the basis of the hypothesis that the expression of ANX4 in CCA is regulated by a unique transcription mechanism, we explored the cis‐elements involved in CCA‐specific ANX4 expression using a luciferase reporter. We compared the transcriptional activities of the region from −1534 to +1010 relative to the ANX4 transcription start site in CCA and non‐CCA‐type cell lines, and found that two repeated binding motifs for the tumor suppressor protein, p53, in the first intron of ANX4 were involved in CCA‐specific transcriptional activity. Furthermore, chromatin immunoprecipitation showed that endogenous p53 bound to this site in CCA cell lines. Moreover, the use of short interference RNA to silence the p53 gene decreased the transcriptional activity and mRNA expression of ANX4 in CCA cell lines. Thus, the ANX4 gene is, at least in part, regulated by p53 in CCA cells. Mutations in the p53 gene were absent and levels of p53 target genes were higher in several CCA‐derived cell lines. Although the expression of ANX4 is typically low in these non‐CCA cell lines, ANX4 levels were elevated more than three‐fold by the overexpression of wild‐type but not mutant p53. Therefore, we conclude that the ANX4 gene is a direct transcriptional target of p53, and its expression is enhanced by wild‐type p53 in CCA cells.

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 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.

Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

Elucidating the androgen‐independent growth mechanism is critical for developing effective treatment strategies to combat androgen‐independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP‐derived androgen‐independent cell line, LNCaP‐AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP‐AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP‐AI cells. Furthermore, pull‐down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation‐mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen‐independent prostate cancer cell growth by changing the interaction partners.

Depressive-Like Behaviors Are Regulated by NOX1/NADPH Oxidase by Redox Modification of NMDA Receptor 1

Involvement of reactive oxygen species (ROS) has been suggested in the development of psychiatric disorders. NOX1 is a nonphagocytic form of NADPH oxidase whose expression in the nervous system is negligible compared with other NOX isoforms. However, NOX1-derived ROS increase inflammatory pain and tolerance to opioid analgesia. To clarify the role of NOX1 in the brain, we examined depressive-like behaviors in mice deficient in Nox1 (Nox1−/Y). Depressive-like behaviors induced by chronic social defeat stress or administration of corticosterone (CORT) were significantly ameliorated in Nox1−/Y. Generation of ROS was significantly elevated in the prefrontal cortex (PFC) of mice administrated with CORT, while NOX1 mRNA was upregulated only in the ventral tegmental area (VTA) among brain areas responsible for emotional behaviors. Delivery of miRNA against NOX1 to VTA restored CORT-induced depressive-like behaviors in wild-type (WT) littermates. Administration of CORT to WT, but not to Nox1−/Y, significantly reduced transcript levels of brain-derived neurotrophic factor (bdnf), with a concomitant increase in DNA methylation of the promoter regions in bdnf. Delivery of miRNA against NOX1 to VTA restored the level of BDNF mRNA in WT PFC. Redox proteome analyses demonstrated that NMDA receptor 1 (NR1) was among the molecules redox regulated by NOX1. In cultured cortical neurons, hydrogen peroxide significantly suppressed NMDA-induced upregulation of BDNF transcripts in NR1-expressing cells but not in cells harboring mutant NR1 (C744A). Together, these findings suggest a key role of NOX1 in depressive-like behaviors through NR1-mediated epigenetic modification of bdnf in the mesoprefrontal projection. SIGNIFICANCE STATEMENT NADPH oxidase is a source of reactive oxygen species (ROS) that have been implicated in the pathogenesis of various neurological disorders. We presently showed the involvement of a nonphagocytic type of NADPH oxidase, NOX1, in major depressive disorders, including behavioral, biochemical, and anatomical changes in mice. The oxidation of NR1 by NOX1-derived ROS was demonstrated in prefrontal cortex (PFC), which may be causally linked to the downregulation of BDNF, promoting depressive-like behaviors. Given that NOX1 is upregulated only in VTA but not in PFC, mesocortical projections appear to play a crucial role in NOX1-dependent depressive-like behaviors. Our study is the first to present the potential molecular mechanism underlying the development of major depression through the NOX1-induced oxidation of NR1 and epigenetic modification of bdnf.

Mutagenesis of longer inserts by the ligation of two PCR fragments amplified with a mutation primer

We report a method for efficient mutagenesis of DNA in large vectors without subcloning. Two segments of the target DNA sequence, one having a mutation introduced via a mutant primer, were amplified by PCR and then the purified fragments were ligated to a vector. The mutation efficiency was nearly 100%.

Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent

2‐DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2‐D gels. To precisely locate spots of immunoreactive proteins in 2‐D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer‐based reagent (N‐102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC‐MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.