Yoko Ino

Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

Nα-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosomes protein synthesis function.

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.

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.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.

Phosphoproteome analysis of Lotus japonicus seeds.

In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase‐specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM‐kinase target motifs, X‐X‐pS/pT‐Q‐X‐X, were detected in cotyledons. Moreover, by real‐time RT‐PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM‐kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 (http://proteomecentral.proteomexchange.org/dataset/PXD000053).

Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post‐translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed.

Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis

Kawasaki disease (KD) is a systemic vasculitis and childhood febrile disease that can lead to cardiovascular complications. The diagnosis of KD depends on its clinical features, and thus it is sometimes difficult to make a definitive diagnosis. In order to identify diagnostic serum biomarkers for KD, we explored serum KD-related proteins, which differentially expressed during the acute and recovery phases of two patients by mass spectrometry (MS). We identified a total of 1,879 proteins by MS-based proteomic analysis. The levels of three of these proteins, namely lipopolysaccharide-binding protein (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute phase patients. In contrast, the level of retinol-binding protein 4 (RBP4) was decreased. To confirm the usefulness of these proteins as biomarkers, we analyzed a total of 270 samples, including those collected from 55 patients with acute phase KD, by using western blot analysis and microarray enzyme-linked immunosorbent assays (ELISAs). Over the course of this experiment, we determined that the expression level of these proteins changes specifically in the acute phase of KD, rather than the recovery phase of KD or other febrile illness. Thus, LRG1 could be used as biomarkers to facilitate KD diagnosis based on clinical features.

Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma

Background There is currently no reliable serum biomarker for ovarian clear cell carcinoma (CCC), a highly lethal histological subtype of epithelial ovarian cancer (EOC). Previously, using a proteome-based approach, we identified tissue factor pathway inhibitor 2 (TFPI2) as a candidate serum biomarker for CCC. In this study, we sought to evaluate the clinical diagnostic performance of TFPI2 in preoperative prediction of CCC. Methods Serum TFPI2 levels were measured in serum samples from a retrospective training set consisting of patients with benign and borderline ovarian tumors, EOC subtypes, and uterine diseases. Via receiver operating characteristic (ROC) analyses, we compared the diagnostic performance of TFPI2 with that of CA125 in discrimination of patients with ovarian CCC from other patient groups. The observed diagnostic performances were examined in a prospective validation set. Results The 268-patient training set included 29 patients with ovarian CCC. Unlike CA125, which was also elevated in patients with endometriosis and several EOC subtypes, serum TFPI2 levels were specifically elevated only in ovarian CCC patients, consistent with the mRNA expression pattern in tumor tissues. The area under the ROC curve (AUC) of serum TFPI2 was obviously higher than that of CA125 for discrimination of CCC from other ovarian diseases (AUC = 0.891 versus 0.595). Applying a cut-off value of 280 pg/mL, TFPI2 could distinguish early-stage (FIGO I and II) CCC from endometriosis with 72.2% sensitivity, 93.3% specificity, and 88.8% accuracy. Similar results were confirmed in an independent 156-patient prospective validation set. Conclusions TFPI2 is a useful serum biomarker for preoperative clinical diagnosis of CCC.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.