282 results on '"Hisashi Hirano"'
Search Results
2. Bone Scan Index (BSI) scoring by using bone scintigraphy and circulating tumor cells (CTCs): predictive factors for enzalutamide effectiveness in patients with castration-resistant prostate cancer and bone metastases
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Hisashi Hirano, Masayoshi Nagata, Naoya Nagaya, So Nakamura, Takeshi Ashizawa, Yan Lu, Haruna Kawano, Kosuke Kitamura, Yoshiro Sakamoto, Kazuhiko Fujita, Hideyuki Isobe, Akira Tsujimura, Satoru Muto, and Shigeo Horie
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Multidisciplinary - Abstract
Reports of Bone Scan Index (BSI) calculations as imaging biomarkers to predict survival in patients with metastatic castration-resistant prostate cancer (mCRPC) have been mainly from retrospective studies. To evaluate the effectiveness of enzalutamide (ENZ) in Japanese patients with mCRPC and bone metastases using BSI (bone scintigraphy) and circulating tumor cell (CTC) analysis. Prospective, single-arm study at Juntendo University affiliated hospitals, Japan. Patients were administered 160 mg ENZ daily, with 3 monthly assessments: BSI, prostate specific antigen (PSA), CTC and androgen receptor splicing variant-7 (AR-V7) status. Primary endpoint: BSI-decreasing rate after ENZ treatment. Secondary endpoints: PSA-decreasing rate and progression free survival (PFS). Statistical analyses included the Wilcoxon t-test, Cox proportional hazard regression analysis, and log-rank test. Median observation period: 17.9 months, and median PFS: 13.8 (2.0–43.9) months (n = 90 patients). A decrease in BSI compared to baseline as best BSI change on ENZ treatment was evident in 69% patients at the end of the observation period (29% patients showed a complete response, BSI 0.00). At 3 months 67% patients showed a ≥ 50% PSA reduction, and 70% after ENZ treatment. PSA decline (3 months) significantly associated with a prolonged median PFS: 18.0 (estimated) versus 6.4 months (HR 2.977 [95% CI 1.53–5.78], p = 0.001). Best BSI decline response significantly associated with a prolonged PFS: 18.1(estimated) versus 7.8 months (HR 2.045 [95% CI: 1.07–3.90], p = 0.029). CTC negative status (n = 20) significantly associated with a prolonged PFS: 13.4 [estimated] vs 8.6 months (HR 2.366, 95% CI 0.97–5.71, p = 0.041). CTC positive/AR-V7 positive status significantly associated with a shorter PFS: 5.9 months (HR 8.56, 95% CI 2.40–30.43, p = 0.0087). -reduction (3 months) and BSI-reduction (on ENZ treatment) were significant response biomarkers, and a negative CTC status was a predictive factor for ENZ efficacy in patients with mCRPC.
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- 2023
3. Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics
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Hisashi Hirano and Jun Shirakawa
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Proteomics ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Electrophoresis, Polyacrylamide Gel ,Phosphorylation ,Phosphoproteins ,Molecular Biology ,Biochemistry ,Phosphates - Abstract
Phosphate-binding tag (Phos-tag) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is an important development capable of analyzing the phosphorylation state of proteins. Conventionally, proteins were separated via SDS-PAGE and Phos-tag SDS-PAGE that use different gels to identify phosphorylated proteins. However, it was often difficult to compare the electrophoretic mobility of the proteins in the different gels used. The recently developed Phos-tag diagonal electrophoresis has been able to solve this problem. It can indicate the SDS-PAGE and Phos-tag SDS-PAGE patterns on a single gel; therefore, phosphorylated proteins can be distinguished easily from non-phosphorylated proteins. This review assesses the importance of Phos-tag electrophoresis, which enables the analysis of protein phosphorylation states, in the field of proteomics. Additionally, this review describes the significance and actual experimental technique of Phos-tag diagonal electrophoresis, which was recently developed to overcome the drawbacks of Phos-tag SDS-PAGE. Although shotgun analysis of proteins allows detecting many phosphorylation sites, it is challenging to clarify the differences in the phosphorylation states of protein molecules using this technique. Therefore, Phos-tag SDS-PAGE is frequently used to determine the phosphorylation state of proteins. This technique has become more powerful with the recent development of Phos-tag diagonal electrophoresis. Abbreviations: BIS, N,N’-methylenebis(acrylamide); CBB, Coomassie brilliant blue R250; ESI, electrospray ionization; hnRNP, heterogeneous ribonucleoprotein K; LTQ–Orbitrap, Linear trap quadrupole–Orbitrap; LC, liquid chromatography; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; Phos-tag, phosphate-binding tag [1,3-bis [bis (pyridine-2-ylmethyl) amino] propane-2-olate]; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF, time of flight; 2D-DIGE, fluorescence-labeled two-dimensional difference gel electrophoresis; 2-DE, two-dimensional gel electrophoresis
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- 2022
4. Efficacy of cabazitaxel and androgen splicing variant-7 status in circulating tumor cells in Asian patients with metastatic castration-resistant prostate cancer
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Takeshi Ashizawa, Masayoshi Nagata, So Nakamura, Hisashi Hirano, Naoya Nagaya, Yan Lu, and Shigeo Horie
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Male ,Prostatic Neoplasms, Castration-Resistant ,Multidisciplinary ,Receptors, Androgen ,Nitriles ,Androgens ,Humans ,Protein Isoforms ,Docetaxel ,Prospective Studies ,Prostate-Specific Antigen ,Neoplastic Cells, Circulating ,Tomography, X-Ray Computed - Abstract
BACKGROUND: Androgen receptor splice variant-7(AR-V7) expression in circulating tumor cells(CTCs) in metastatic castration-resistant prostate cancer (mCRPC) is associated with resistance to abiraterone and enzalutamide. We had objectives to determine whether cabazitaxel(CBZ) was equally effective in AR-V7 positive and negative CRPC, and if AR-V7 positive patients retained CBZ sensitivity.METHODS: This study was first-of-a-kind, Asian validation, prospective, open-label study: CBZ in Japanese mCRPC patients after docetaxel. 48 patients completed 4 CBZ cycles (recruited: 2017–2020, Juntendo University Hospitals). Primary endpoint was prostate-specific antigen response rate(PSA-RR), and secondary endpoints were overall survival(OS), Bone Scan Index[BSI]-change rate of bone metastases by bone scintigraphy, and safety assessments. PSA-RR(≥50% decline from baseline) for: CTC-/ARV7-, CTC+/ARV7-, CTC+/AR+ groups. RESULTS: PSA-RR shown ≥30% was 38%(18/48) and ≥50% was 26%(12/48). BSI-change rate shown ≥-30% was 19%(9/41) and ≥-50% was 17%(8/41). Median OS was 13.7(12.2–18.9) months. PSA decline in early CBZ treatment associated with OS(p=0.00173). BSI decline associated with OS(p=0.0194). PSA-RR(≥50%) was 21%(3/14) in CTC-/ARV7- group, 16%(4/25) in CTC+/ARV7- and 12%(1/8) in CTC+/ARV7+. No statically significant differences between groups. AR-V7 in CTCs at baseline not associated with OS. CONCLUSIONS: AR-V7 in CTCs was not associated with resistance to CBZ. BSI and PSA reducing responses in early CBZ may predict OS.
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- 2022
5. Effects of microgravity exposure and fructo-oligosaccharide ingestion on the proteome of soleus and extensor digitorum longus muscles in developing mice
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Hisashi Hirano, Yoshinobu Ohira, Ayuko Kimura, Yoichi Kurata, Takashi Ohira, Satoshi Furukawa, Hiroyuki Kagawa, Yoko Ino, Yayoi Kimura, Kenji Egashira, Yusuke Nakai, Mitsuo Kimura, and Chie Matsuda
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medicine.medical_specialty ,Cell biology ,Physics and Astronomy (miscellaneous) ,Physiology ,Materials Science (miscellaneous) ,Medicine (miscellaneous) ,Oxidative phosphorylation ,Spaceflight ,medicine.disease_cause ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Article ,law.invention ,chemistry.chemical_compound ,law ,Internal medicine ,Detoxification ,medicine ,Ingestion ,QP1-981 ,chemistry.chemical_classification ,Preventive medicine ,Glutathione ,Oligosaccharide ,Agricultural and Biological Sciences (miscellaneous) ,Endocrinology ,chemistry ,Space and Planetary Science ,Fermentation ,Oxidative stress ,TP248.13-248.65 ,Biotechnology - Abstract
著者人数: 13名, 形態: カラー図版あり, Number of authors: 13, Physical characteristics: Original contains color illustrations, Accepted: 2021-08-26, 資料番号: PA2210082000
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- 2021
6. The Use of Urine Mycobacterium tuberculosis Complex Polymerase Chain Reaction as a Predictive Factor for Recurrence and Progression After Intravesical Bacillus Calmette-Guérin Therapy in Patients with Non–muscle‑invasive Bladder Cancer
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Takeshi Ashizawa, Shigeo Horie, Masayoshi Nagata, Keisuke Saito, Kousuke Kitamura, Hisamitsu Ide, Satoru Muto, Yan Lu, Shuji Isotani, Hiroki Koyasu, Hisashi Hirano, Raizo Yamaguchi, and Yasuhiro Noma
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medicine.medical_specialty ,Mycobacterium tuberculosis complex polymerase chain reaction ,Urology ,Gastroenterology ,Non–muscle invasive ,Maintenance therapy ,Internal medicine ,Bacillus Calmette-Guérin ,medicine ,Survival analysis ,RC254-282 ,Predictive marker ,Bladder cancer ,biology ,Surrogate endpoint ,business.industry ,Standard treatment ,Hazard ratio ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,biology.organism_classification ,medicine.disease ,Diseases of the genitourinary system. Urology ,Mycobacterium tuberculosis complex ,RC870-923 ,business - Abstract
Background Intravesical bacillus Calmette-Guérin (BCG) instillation is a standard treatment for non–muscle-invasive bladder cancer (NMIBC); however, not all patients benefit from BCG therapy. Currently, no surrogate marker exists to predict BCG efficacy, and thereby, identify patients who will benefit from this treatment. Objective To evaluate the utility of urine Mycobacterium tuberculosis complex polymerase chain reaction (MTC-PCR) assay as a predictive marker for recurrence and progression following BCG therapy. Design, setting, and participants A prospective analysis was carried out for of intermediate- or high-risk NMIBC patients who received BCG instillation for the first time. Urine samples, for MTC-PCR assay, were collected at baseline and annually for up to 10 yr after the last BCG instillation, including induction and maintenance therapy. The first postoperative sample for MTC-PCR was taken at 1 yr from the last instillation. Outcome measurements and statistical analysis A survival analysis was performed using the Kaplan-Meier method, and risk factors for recurrence and progression after BCG treatment were assessed using Cox regression analysis. Results and limitations During follow-up (median: 57 mo), 468/521 samples (89.8%) were MTC-PCR positive, and 108/123 patients (87.8%) exhibited MTC-PCR positivity at least once. Five-year recurrence- and progression-free survival in patients who were not MTC-PCR positive was significantly lower than in patients who were MTC-PCR positive at least once (p, Take Home Message In intermediate- or high-risk non–muscle-invasive bladder cancer patients after intravesical bacillus Calmette-Guérin (BCG) therapy, Mycobacterium tuberculosis complex polymerase chain reaction (MTC-PCR) positivity at least once was a significant prognostic factor in recurrence. A new adequate biomarker, urine MTC-PCR, is very useful noninvasive surrogate marker to predict the recurrence after BCG therapy.
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- 2021
7. Phos-tag diagonal electrophoresis to analyze phosphoproteins
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Hisashi Hirano and Jun Shirakawa
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Electrophoresis ,Chromatography ,biology ,Chemistry ,Diagonal ,Phos ,biology.organism_classification - Published
- 2021
8. Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis
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Yoko Ino, Eiji Kinoshita, Emiko Kinoshita‐Kikuta, Tomoko Akiyama, Yusuke Nakai, Kohei Nishino, Makoto Osada, Akihide Ryo, Hisashi Hirano, Tohru Koike, and Yayoi Kimura
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Phosphopeptides ,Proteomics ,Titanium ,Phosphorylation ,Molecular Biology ,Biochemistry ,Chromatography, Affinity ,Mass Spectrometry - Abstract
Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO
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- 2021
9. Clinical use of expanded prostate cancer index composite-based health-related quality of life outcomes after robot-assisted radical prostatectomy for localized prostate cancer
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Shigeo Horie, Masayoshi Nagata, Kazuhiro Fukuda, Takeshi Ashizawa, Hisashi Hirano, Satoru Muto, Kosuke Kitamura, Yasuhiro Noma, Hiroki Koyasu, Fumitaka Shimizu, Toshiyuki China, and Shuji Isotani
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medicine.medical_specialty ,business.industry ,Prostatectomy ,Urology ,Urinary system ,medicine.medical_treatment ,Expanded Prostate Cancer Index Composite ,Urinary incontinence ,medicine.disease ,Prostate cancer ,Quality of life ,Interquartile range ,medicine ,Analysis of variance ,medicine.symptom ,business - Abstract
Background This study aimed to assess the longitudinal health-related quality of life (HRQOL) using the Expanded Prostate Cancer Index Composite (EPIC) and HRQOL change between the nerve-sparing technique in Japanese men treated with robot-assisted radical prostatectomy (RARP). Methods A total of 573 patients who received RARP were included in this study. EPIC questionnaire was administered before treatment and up to 36 months after RARP. Clinical recovery was defined as half of the standard deviation of the baseline score for each domain. We divided all patients into recovery group or nonrecovery group. The time from survey to each domain recovery was calculated using the Kaplan–Meier method. We compared the sexual and urinary score change between groups using analysis of variance to confirm the effect of nerve-sparing technique. Results The median age was 67 years (interquartile range, 62–71 years). The mean score of all urinary domains worsened noticeably after 1 month. All postoperative urinary summary, function, and incontinence scores were significantly lower than preoperative scores up to 3 years post-RARP. Postoperative sexual summary and functional scores were significantly lower than preoperative score at all follow-up times throughout the 36 months. The recovery rate for the urinary incontinence domain was the lowest (44.5%), whereas the recovery rate for the urinary irritative–obstructive domain was the highest (73.7%). In the sexual domain, the bother domain had a higher recovery rate (73.0%) than the functional domain (29.7%). Although the recovery of sexual domains was slower compared with other domains, by 36 months after RARP, almost all values had recovered. Compared with other technique groups, bilateral intrafascial nerve-sparing group showed significantly decreased change in subscale scores before and after RARP in several sexual and urinary domain. Conclusion The time course and extent of functional and bother domain recovery documented in this study may prove useful for RARP patient selection in Japan.
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- 2021
10. Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome
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Youngsoo Kim, Seon Young Choi, Hye Jung Kim, Su-Jin Lee, Je-Yoel Cho, Ji Hyun Back, Won Suk Yang, Younghee Ahn, Eunok Paek, Prashant Kaushal, Seong Jun Park, Eugene C. Yi, Jin Young Kim, Seonjeong Lee, Seungjin Na, Shinyeong Ju, Hee-Sung Ahn, Yumi Kwon, Yae Eun Park, Kang Sik Park, Ji Eun Lee, Mohammad Humayun Kabir, Sung Ho Shin, Jin-Won Lee, Hisashi Hirano, Jeonghun Yeom, J. Eugene Lee, Chul-Won Ha, Kwang Pyo Kim, Hyun Gyo Jung, Un Beom Kang, Jihye Shin, Oh Young Bang, Cheolju Lee, Eun-Hee Kim, and Eun Young Hong
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Proteomics ,Serum ,0301 basic medicine ,Proteome ,Cell ,Computational biology ,Biochemistry ,Extracellular vesicles ,Mass Spectrometry ,03 medical and health sciences ,Stable isotope labeling by amino acids in cell culture ,medicine ,Animals ,Humans ,Database search engine ,Databases, Protein ,Cells, Cultured ,030102 biochemistry & molecular biology ,Chemistry ,General Chemistry ,Mass spectrometric ,Culture Media ,030104 developmental biology ,medicine.anatomical_structure ,Reference database ,Cattle - Abstract
We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.
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- 2019
11. Proteome and behavioral alterations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice
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Toshio Ohshima, Tetsuya Asano, Haruko Nakamura, Hisashi Hirano, Yuko Kawamoto, Yayoi Kimura, Fumiaki Tanaka, Yoshio Goshima, Naoya Yamashita, Jun Nakabayashi, Fumio Nakamura, Hiroko Makihara, and Aoi Takahashi-Jitsuki
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0301 basic medicine ,Proteome ,Mice, Transgenic ,Nerve Tissue Proteins ,Biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Semaphorin ,Gene knockin ,Animals ,Phosphorylation ,Neurons ,Glycogen Synthase Kinase 3 beta ,Behavior, Animal ,Kinase ,Cyclin-dependent kinase 5 ,Cyclin-Dependent Kinase 5 ,Neurofibrillary Tangles ,Semaphorin-3A ,SEMA3A ,Cell Biology ,Cell biology ,030104 developmental biology ,Mutation ,Intercellular Signaling Peptides and Proteins ,Collapsin response mediator protein family ,Signal transduction ,030217 neurology & neurosurgery - Abstract
CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3β (GSK-3β) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2ki/ki) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2ki/ki mice. The crmp2ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain.
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- 2018
12. Basic 7S globulin in plants
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Hisashi Hirano
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0301 basic medicine ,Microorganism ,Biophysics ,7s globulin ,Peptide ,Biology ,Biochemistry ,03 medical and health sciences ,Homologous chromosome ,Storage protein ,Plant Proteins ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,fungi ,Seed Storage Proteins ,food and beverages ,Globulins ,030104 developmental biology ,chemistry ,Cancer cell ,Seeds ,Soybeans ,Antibacterial activity ,Carrier Proteins ,Hormone - Abstract
Soybean seed basic 7S globulin (Bg7S)-like proteins are found in many plant species. Bg7S was originally thought to be a major seed storage protein but was later found to be multifunctional, with stress response, antibacterial activity, hormone receptor-like activity. Moreover, functional differences between Bg7S proteins from legumes and other plants have been revealed. In non-leguminous plants, Bg7S molecules inhibit the invasion of pathogenic microorganisms. However, although leguminous plants have a peptide called leg-insulin that can bind to Bg7S, non-leguminous plants do not have leginsulin. Bg7S in leguminous plants and other plants may have evolved in functionally different directions. Several homologs of Bg7S in plants are reported, but there is no homolog of this protein in peas, suggesting that the pea evolution might have followed a different route when compared to other leguminous plants. Although the functions of Bg7S are well documented in plants, recent studies suggest that this protein is also important in controlling blood glucose level, blood pressure and plasma cholesterol level, and cancer cell antiproliferative actions.
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- 2021
13. Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma
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Ren Takahashi, Yuki Kawata, Natsumi Sato, Hisashi Hirano, Hirokazu Kimura, Yukio Morita, Ayuko Kimura, Yukari Miyano, and Kiyotaka Fujita
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0301 basic medicine ,Immunoglobulin A ,Dimer ,In silico ,Serum Albumin, Human ,Article ,Catalysis ,Inorganic Chemistry ,lcsh:Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,docking simulation ,Glycation ,Hyperviscosity syndrome ,medicine ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,Multiple myeloma ,Aged ,IgA-albumin complex ,mass spectrometry ,Aged, 80 and over ,biology ,Organic Chemistry ,Albumin ,General Medicine ,Middle Aged ,medicine.disease ,J chain ,Computer Science Applications ,Molecular Docking Simulation ,multiple myeloma ,030104 developmental biology ,chemistry ,Biochemistry ,lcsh:Biology (General) ,lcsh:QD1-999 ,030220 oncology & carcinogenesis ,biology.protein ,Protein Multimerization ,oxidized albumin ,Oxidation-Reduction ,Protein Binding - Abstract
Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein–protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrometry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera. Molecular interaction analyses in silico implied that dimeric IgA and albumin interacted not only via disulfide bond formation, but also via noncovalent bonds. Disulfide bonds were predicted between Cys34 of albumin and Cys311 of IgA, resulting in an oxidized form of albumin. Furthermore, complex formation prolongs the half-life of IgA molecules in the IgA-albumin complex, leading to excessive glycation of IgA molecules and affects the accumulation of IgA in serum. These findings may demonstrate why complications such as hyperviscosity syndrome occur more often in patients with IgA dimer producing multiple myeloma.
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- 2021
14. Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE
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Natsumi Sato, Makoto Osada, Takao Kawakami, Eiji Kinoshita, Norihisa Ootake, Yuki Okawara, Yuriko Hayashi, Ayuko Kimura, Hisashi Hirano, and Kiyotaka Fujita
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0301 basic medicine ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Pyridines ,Diagonal ,Biophysics ,Mass spectrometry ,Phosphoproteins ,Biochemistry ,Yeast ,03 medical and health sciences ,Electrophoresis ,Ovalbumin ,030104 developmental biology ,Proteasome ,biology.protein ,Phosphorylation ,Humans ,Electrophoresis, Polyacrylamide Gel ,Polyacrylamide gel electrophoresis - Abstract
Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and β-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SIGNIFICANCE: Previously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins. : Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome.
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- 2020
15. Linagliptin ameliorates hepatic steatosis via non-canonical mechanisms in mice treated with a dual inhibitor of insulin receptor and IGF-1 receptor
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Yasuo Terauchi, Mayu Kyohara, Yoko Ino, Jinghe Li, Kazuki Tajima, Rie Takayanagi, Hisashi Hirano, Ryota Inoue, Nozomi Goto, Yu Togashi, Tomoko Okuyama, Taiga Ichikawa, Heidrun Vethe, Jun Shirakawa, Haruka Ohnuma, Shingo Yamasaki, Yayoi Kimura, and Daisuke Miyashita
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Blood Glucose ,Male ,0301 basic medicine ,Linsitinib ,Receptor, IGF Type 1 ,lcsh:Chemistry ,chemistry.chemical_compound ,0302 clinical medicine ,Non-alcoholic Fatty Liver Disease ,insulin resistance ,Insulin ,lcsh:QH301-705.5 ,Spectroscopy ,diabetes ,biology ,hepatic steatosis ,Fatty liver ,Imidazoles ,phosphoproteomics ,General Medicine ,Computer Science Applications ,Pyrazines ,Lipogenesis ,Sirtuin ,Intercellular Signaling Peptides and Proteins ,Aryl Hydrocarbon Hydroxylases ,medicine.drug ,medicine.medical_specialty ,Linagliptin ,030209 endocrinology & metabolism ,Perilipin-2 ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,proteomics ,Insulin resistance ,DPP-4 inhibitors ,Internal medicine ,medicine ,Animals ,Physical and Theoretical Chemistry ,Cytochrome P450 Family 2 ,insulin signaling ,Molecular Biology ,Triglycerides ,Insulin-like growth factor 1 receptor ,Organic Chemistry ,medicine.disease ,Receptor, Insulin ,Mice, Inbred C57BL ,Insulin receptor ,030104 developmental biology ,Endocrinology ,Gene Expression Regulation ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,Steroid Hydroxylases ,Hepatocytes ,biology.protein ,Steatosis - Abstract
Background and Purpose: Abnormal hepatic insulin signaling is a cause or consequence of hepatic steatosis. DPP-4 inhibitors might be protective against fatty liver. We previously reported that the systemic inhibition of insulin receptor (IR) and IGF-1 receptor (IGF1R) by the administration of OSI-906 (linsitinib), a dual IR/IGF1R inhibitor, induced glucose intolerance, hepatic steatosis, and lipoatrophy in mice. In the present study, we investigated the effects of a DPP-4 inhibitor, linagliptin, on hepatic steatosis in OSI-906-treated mice. Experimental Approach: We treated C57BL/6J male mice either with vehicle, linagliptin, OSI-906 or OSI-906 + linagliptin for 7 days. We also conducted proteomic and phosphoproteomic analyses of the liver from those mice. Key Results: Unlike high-fat diet-induced hepatic steatosis, OSI-906-induced hepatic steatosis is not characterized by elevations in inflammatory responses or oxidative stress levels. Linagliptin improved OSI-906-induced hepatic steatosis via an insulin-signaling-independent pathway, without altering glucose levels, free fatty acid levels, gluconeogenic gene expressions in the liver, or visceral fat atrophy. Hepatic quantitative proteomic and phosphoproteomic analyses revealed that perilipin-2 (PLIN2), major urinary protein 20 (MUP20), cytochrome P450 2b10 (CYP2B10), nicotinamide N-methyltransferase (NNMT), and sirtuin families are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Conclusion and Implications: Linagliptin improved hepatic steatosis induced by IR and IGF1R inhibition via a previously unknown mechanism that did not involve gluconeogenesis, lipogenesis, or inflammation, suggesting the non-canonical actions of DPP-4 inhibitors in the treatment of hepatic steatosis under insulin-resistant conditions.
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- 2020
16. TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes
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Tetsuya Kotani, Yu Oikawa, Yoshinori Ohsumi, Hiromi Kirisako, Yayoi Kimura, Hisashi Hirano, Hitoshi Nakatogawa, and Yui Tomioka
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Nucleophagy ,Immunoelectron microscopy ,ATG8 ,Autophagy ,Cell Biology ,mTORC1 ,Biology ,Cell biology ,Cell Death and Autophagy ,Nuclear Pore Complex Proteins ,stomatognathic diseases ,Report ,otorhinolaryngologic diseases ,Nuclear Pore ,Nucleoporin ,Signal transduction ,Nuclear pore - Abstract
Autophagy selectively degrades a wide range of cellular components to regulate cellular functions or maintain cellular homeostasis. Tomioka et al. reveal that the nuclear pore complex and nucleoporins are degraded by selective autophagy upon inactivation of Tor kinase complex 1 in Saccharomyces cerevisiae., The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor–binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope–derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term “NPC-phagy” and “nucleoporinophagy.”
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- 2020
17. Impact of Pretreatment Total Cholesterol Level Is Associated With Metastasis of Prostate Cancer
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Yan Lu, Shigeo Horie, Hisamitsu Ide, Yasuyuki Inoue, Hisashi Hirano, and Hiroshi Okada
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Oncology ,Adult ,Male ,medicine.medical_specialty ,Health (social science) ,030232 urology & nephrology ,lcsh:Medicine ,Metastasis ,03 medical and health sciences ,Prostate cancer ,0302 clinical medicine ,Risk Factors ,Diabetes mellitus ,Internal medicine ,medicine ,metastasis ,Humans ,Testosterone ,Risk factor ,Neoplasm Metastasis ,Original Research ,Aged ,Aged, 80 and over ,hypercholesterolemia ,business.industry ,lcsh:R ,Public Health, Environmental and Occupational Health ,Prostatic Neoplasms ,Arteriosclerosis ,Middle Aged ,Prostate-Specific Antigen ,medicine.disease ,prostate cancer ,Prostatic Disorders ,030220 oncology & carcinogenesis ,Multivariate Analysis ,Metabolic syndrome ,business ,Body mass index - Abstract
Metabolic syndrome is reported to play a role in the genesis and development not only of angina, arteriosclerosis, diabetes, and osteoporosis but also of prostate cancer. Hypercholesterolemia is a strong risk factor in prostate cancer development. The current study was conducted to analyze whether pretreatment serum levels of cholesterol correlate with prostate cancer metastasis. Three hundred fifty-one subjects who received a histopathological diagnosis of prostate cancer were evaluated by clinical factors such as age, body mass index (BMI), disease stage, Gleason score, prostate-specific antigen (PSA), total cholesterol, Luteinizing hormone (LH), testosterone, and free testosterone. A multivariate analysis was performed on these factors, and a statistically significant difference was identified in total cholesterol level ( p =.01) and PSA ( p < .001). The total cholesterol level was higher in cases of metastatic prostate cancer compared to nonmetastatic prostate cancer in this study and therefore may be a predictive factor for poor prognosis.
- Published
- 2020
18. Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity
- Author
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Masa Aki Nakaya, Hisashi Hirano, Yohei Yoshihama, Miho Shimada, Kazunori Sasaki, Hidehisa Takahashi, Akio Yamashita, Noriko Kojitani, Hiroko Hirose, Hidefumi Suzuki, Shigeo Ohno, and Ayumi Takayanagi
- Subjects
0301 basic medicine ,Gene isoform ,Male ,Polarity (physics) ,Telomere-Binding Proteins ,Cell Cycle Proteins ,Nerve Tissue Proteins ,General Biochemistry, Genetics and Molecular Biology ,Shelterin Complex ,Cell Line ,Tight Junctions ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Dogs ,Cell polarity ,Animals ,Humans ,Small GTPase ,lcsh:QH301-705.5 ,Protein Kinase C ,Epithelial polarity ,Adaptor Proteins, Signal Transducing ,Tight junction ,Chemistry ,Cell Polarity ,Epithelial Cells ,Cell biology ,030104 developmental biology ,HEK293 Cells ,lcsh:Biology (General) ,MCF-7 Cells ,Ankyrin repeat ,Rap1 ,Female ,Caco-2 Cells ,Carrier Proteins ,Cell Adhesion Molecules ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Summary: Epithelial cells establish apicobasal polarity by forming tight junctions (TJs) at the apical-lateral boundary, which play fundamental roles in physiological functions. An evolutionarily conserved atypical protein kinase C (aPKC)-partitioning defective (PAR) complex functions as a platform for TJ assembly during cell polarity establishment. However, how this complex converts the spatial cues into a subsequent active unit is unclear. Here, we identify an epithelial isoform of Shank2 as a mediator of the aPKC-PAR complex. Shank2 binds to and colocalizes with aPKC at apical junctional regions of polarized epithelial cells. Shank2 knockdown results in defects in TJ formation. Mechanistically, we find that the N-terminal SPN domain is required for the junctional localization of Shank2 and binds to the active form of Rap1 small GTPase, which is involved in TJ formation. Our findings suggest that a close physical and functional relationship between aPKC and Shank2-active Rap1 signaling serves as the platform for TJ assembly to regulate epithelial cell polarity. : Sasaki et al. demonstrate that epithelial Shank2 localizes at tight junctions (TJs) via its epithelia-specific N terminus and interacts with aPKC. Through small GTPase Rap1 signaling, the aPKC-Shank2 complex functions as a platform for TJ assembly during the establishment of the epithelial cell polarity. Keywords: cell polarity, tight junction, aPKC-PAR complex, Shank2, Rap1, SPN domain, ankyrin repeats
- Published
- 2020
19. Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice
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Kyoko Hiramatsu, Hisashi Hirano, Hiroyuki Kagawa, Yoko Ino, Hironobu Morita, Yayoi Kimura, Kenji Egashira, Mitsuo Kimura, Ayuko Kimura, Yusuke Nakai, Yoichi Kurata, Shun-suke Moriya, Masao Kawakita, and Takashi Ohira
- Subjects
0301 basic medicine ,Proteomics ,Spermine oxidase ,Biophysics ,Spermine ,Hypergravity ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Tandem Mass Spectrometry ,medicine ,Animals ,Muscle, Skeletal ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Skeletal muscle ,musculoskeletal system ,Muscle atrophy ,Cell biology ,Spermidine ,030104 developmental biology ,medicine.anatomical_structure ,Muscle Fibers, Slow-Twitch ,Muscle Fibers, Fast-Twitch ,biology.protein ,medicine.symptom ,Spermidine synthase ,Polyamine ,Chromatography, Liquid ,Muscle Contraction - Abstract
Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. Significance The skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.
- Published
- 2019
20. Protein fractionation for proteomics using the SAINOME-plate
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Sakura Ito, Yusuke Nakai, Tomoko Akiyama, Hisashi Hirano, Makiko Kawamura, Yoko Ino, Yayoi Kimura, Hiroyuki Kagawa, and Masahiro Shimoda
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Chromatography ,Chemistry ,In-gel digestion ,Fractionation ,Proteomics ,Mass spectrometry - Published
- 2018
21. MZB1 in borderline resectable pancreatic cancer resected after neoadjuvant chemoradiotherapy
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Kentaro Miyake, Itaru Endo, Ryutaro Mori, Takashi Murakami, Ryusei Matsuyama, Yuki Homma, Hisashi Hirano, and Akiko Okayama
- Subjects
Male ,Proteomics ,0301 basic medicine ,Oncology ,medicine.medical_specialty ,Stromal cell ,chemical and pharmacologic phenomena ,CD8-Positive T-Lymphocytes ,Lesion ,03 medical and health sciences ,0302 clinical medicine ,Japan ,Stroma ,Internal medicine ,Pancreatic cancer ,medicine ,Humans ,Adaptor Proteins, Signal Transducing ,Aged ,business.industry ,hemic and immune systems ,Chemoradiotherapy, Adjuvant ,Middle Aged ,Marginal zone ,medicine.disease ,Immunohistochemistry ,Neoadjuvant Therapy ,Pancreatic Neoplasms ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cytokines ,Biomarker (medicine) ,Female ,Surgery ,medicine.symptom ,business ,Biomarkers ,CD8 ,Carcinoma, Pancreatic Ductal - Abstract
Background A high accumulation of CD8+ tumor-infiltrating lymphocytes (TILs) induced by neoadjuvant chemoradiotherapy (NACRT) is associated with a favorable prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). However, the correlation between a high accumulation of CD8+ TILs and a favorable prognosis has yet to be fully clarified. The aim of this study was to determine predictive markers of a high accumulation of CD8+ TILs, with a favorable prognosis, using proteomic analysis. Materials and methods We studied 72 resected borderline resectable PDAC patients treated with NACRT between April 2009 and March 2014. Three matched pairs of high CD8+ TIL patients with a favorable prognosis and low CD8+ TIL patients with a poor prognosis were selected. Shotgun proteomics of the stroma and cancerous lesion was performed using formalin-fixed, paraffin-embedded tissue. Validation of the identified proteins was performed using immunohistochemical staining. Relationships between the identified proteins and TILs and clinical outcomes were assessed. Results Marginal zone B- and B1-cell-specific protein (MZB1) was detected in the tumor stroma. MZB1 expression was positively correlated with a high accumulation of CD8+ TILs. High stromal MZB1 expression also correlated with disease-free and overall survival. In a subgroup analysis of CD8+ expression, there was a significant association between stromal MZB1 expression and disease-free and overall survival in the high CD8+ TIL group. Conclusions MZB1 is a potential marker of a high accumulation of CD8+ TILs in borderline resectable PDACs resected after NACRT. Combination of CD8+ TILs with MZB1 may be a new biomarker of resected cases after NACRT.
- Published
- 2017
22. Phosphorylation of Ser1452 on BRG1 inhibits the function of the SWI/SNF complex in chromatin activation
- Author
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Hiroyuki Kagawa, Hisashi Hirano, Ayuko Kimura, Yayoi Kimura, and Noriaki Arakawa
- Subjects
biology ,SWI/SNF complex ,Chemistry ,cells ,genetic processes ,Biophysics ,macromolecular substances ,Biochemistry ,Chromatin remodeling ,Chromatin ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Histone ,Downregulation and upregulation ,Histone methylation ,biology.protein ,Demethylase ,Phosphorylation ,biological phenomena, cell phenomena, and immunity - Abstract
BRG1, one of core subunits of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancers. Previously, we reported significant downregulation of the phosphorylation level of BRG1 on Ser, (, D), or non-phosphorylatable (brg1-S, A) BRG1. Quantitative proteomic analyses revealed upregulation of proteins and phosphoproteins related to linker histone H1s, histone methylation, and protein ubiquitylation in brg1-S, D cells, which may coordinately promote the chromatin inactivation and ubiquitin-dependent degradation of target proteins. Consistent with these results, brg1-S, D cells exhibited an increase in condensed chromatin and polyubiquitylated proteins. In brg1-S, D cells, we also detected downregulation of various cancer-related proteins (e.g., EGFR and MET) as well as decreased migration, proliferation, and sensitivity to taxanes and oxaliplatin. Together, our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins. SIGNIFICANCE: For the first time we demonstrated that the mutation on Ser, phosphorylation site of BRG1, a component of SWI/SNF chromatin remodeling complex, changed protein and phosphoprotein levels of linker histone H1s, binding competitor of histone H1s, and histone methylase/demethylase involved in the heterochromatic histone modifications to promote the chromatin inactivation. In phosphorylation-mimic mutant, significant decrease of various cancer-related proteins as well as migration, proliferation, and sensitivity to specific antitumor agents were detected. Our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins.
- Published
- 2021
23. A Phos-tag-based micropipette-tip method for rapid and selective enrichment of phosphopeptides
- Author
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Eiji Kinoshita, Tohru Koike, Elena Tianfei Yuan, Hisashi Hirano, Maho Kawaguchi, Yayoi Kimura, Emiko Kinoshita-Kikuta, and Yoko Ino
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Aqueous solution ,Chromatography ,Chemistry ,Elution ,Phosphopeptide ,010401 analytical chemistry ,Clinical Biochemistry ,Pipette ,Peptide ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Affinity chromatography ,Agarose ,Selectivity - Abstract
Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
- Published
- 2017
24. Phos-tag World: The path to the future of electrophoresis, pioneered by the Phos-tag
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Yayoi Kimura, Hisashi Hirano, and Yoko Ino
- Subjects
Electrophoresis ,biology ,Chemistry ,Phos ,Path (graph theory) ,Computational biology ,biology.organism_classification ,Proteomics ,Molecular biology - Published
- 2017
25. ModProt: A database for integrating laboratory and literature data concerning protein post-translational modifications
- Author
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Hisashi Hirano, Yayoi Kimura, and Tosifusa Toda
- Subjects
Information retrieval ,Computer science ,Posttranslational modification - Published
- 2017
26. Proteomic analysis of exosome-enriched fractions derived from cerebrospinal fluid of amyotrophic lateral sclerosis patients
- Author
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Noriko Hayashi, Hisashi Hirano, Keita Takahashi, Yoshitoshi Atobe, Mikiko Tada, Fumiaki Tanaka, Haruko Nakamura, Kengo Funakoshi, Hideyuki Takeuchi, Atsuko Katsumoto, Kenichi Tanaka, Yayoi Kimura, Yoichi Kurata, Hiroyuki Kagawa, Shigeru Koyano, Hiroshi Doi, and Misako Kunii
- Subjects
0301 basic medicine ,Proteomics ,Exosomes ,Exosome ,Pathogenesis ,03 medical and health sciences ,0302 clinical medicine ,Cerebrospinal fluid ,medicine ,Humans ,Amyotrophic lateral sclerosis ,Motor Neurons ,Chemistry ,General Neuroscience ,Amyotrophic Lateral Sclerosis ,General Medicine ,medicine.disease ,Molecular biology ,Microvesicles ,030104 developmental biology ,medicine.anatomical_structure ,Immunohistochemistry ,Nucleus ,030217 neurology & neurosurgery ,Function (biology) ,Biomarkers - Abstract
Exosomes contain many proteins associated with neurodegenerative diseases. To identify new candidate biomarkers and proteins associated with amyotrophic lateral sclerosis (ALS), we performed liquid chromatography-tandem mass spectrometry proteomic analysis of exosome-enriched fractions isolated from cerebrospinal fluid (CSF) of sporadic ALS patients using gel filtration chromatography. Proteomic data revealed that three proteins were increased and 11 proteins were decreased in ALS patients. The protein with the greatest increase in exosome-enriched fractions of CSF derived from ALS was novel INHAT repressor (NIR), which is closely associated with nucleolar function. By immunohistochemical analysis, we found that NIR was reduced in the nucleus of motor neurons in ALS patients. Our results demonstrate the potential utility of our methodology for proteomic analysis of CSF exosomes and suggest that nucleolar stress might play a role in sporadic ALS pathogenesis through the dysfunction of NIR.
- Published
- 2019
27. Author response: Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure
- Author
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Hisashi Hirano, Tetsuya Kotani, Yayoi Kimura, Yu Oikawa, Yoshinori Ohsumi, Kumi Harada, Hitoshi Nakatogawa, Machiko Sakoh-Nakatogawa, Hiromi Kirisako, and Hayashi Yamamoto
- Subjects
Autophagy ,Biology ,Pre-autophagosomal structure ,Cell biology - Published
- 2019
28. Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure
- Author
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Hiromi Kirisako, Yoshinori Ohsumi, Hayashi Yamamoto, Kumi Harada, Yayoi Kimura, Hisashi Hirano, Yu Oikawa, Hitoshi Nakatogawa, Tetsuya Kotani, and Machiko Sakoh-Nakatogawa
- Subjects
Autophagosome ,autophagy ,Saccharomyces cerevisiae Proteins ,QH301-705.5 ,ATG8 ,Science ,autophagosome ,S. cerevisiae ,Autophagy-Related Proteins ,ubiquitin-like protein ,Lipid-anchored protein ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,Autophagy-Related Protein 5 ,ATG12 ,chemistry.chemical_compound ,Endopeptidases ,Phosphatidylinositol ,Biology (General) ,Pre-autophagosomal structure ,Phosphatidylethanolamine ,General Immunology and Microbiology ,Chemistry ,General Neuroscience ,Autophagy ,Autophagosomes ,General Medicine ,Cell Biology ,Cell biology ,Protein Transport ,Multiprotein Complexes ,Medicine ,Atg protein ,Protein Kinases ,Autophagy-Related Protein 12 ,Gene Deletion ,Research Article ,Protein Binding - Abstract
In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.
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- 2019
29. The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes
- Author
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Daisuke Noshiro, Hayashi Yamamoto, Hisashi Hirano, Nobuo N. Noda, Chika Kondo-Kakuta, Yuko Fujioka, Yoshinori Ohsumi, Toshio Ando, Yayoi Kimura, Hironori Suzuki, and Sho W. Suzuki
- Subjects
0301 basic medicine ,Atg1 ,Saccharomyces cerevisiae Proteins ,Protein Conformation ,Supramolecular chemistry ,Autophagy-Related Proteins ,Saccharomyces cerevisiae ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Supramolecular assembly ,03 medical and health sciences ,Phagosomes ,Autophagy ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,030102 biochemistry & molecular biology ,Sequence Homology, Amino Acid ,Autophosphorylation ,Membrane Proteins ,Cell Biology ,Autophagy-related protein 13 ,Transport protein ,Cell biology ,Intrinsically Disordered Proteins ,030104 developmental biology ,Multiprotein Complexes ,Protein Kinases ,Developmental Biology ,Protein Binding - Abstract
Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
- Published
- 2016
30. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines
- Author
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Hisashi Hirano, Yusuke Masuishi, Yayoi Kimura, and Noriaki Arakawa
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Multidisciplinary ,Peptide ,Computational biology ,Biology ,Proteomics ,lcsh:Computer applications to medicine. Medical informatics ,Gpi anchored protein ,Cell biology ,carbohydrates (lipids) ,03 medical and health sciences ,030104 developmental biology ,Affinity chromatography ,chemistry ,Cell culture ,lcsh:R858-859.7 ,Identification (biology) ,lipids (amino acids, peptides, and proteins) ,Cancer cell lines ,lcsh:Science (General) ,Lipid raft ,Data Article ,lcsh:Q1-390 - Abstract
We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled “Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment” (Masuishi et al., 2016) [1].
- Published
- 2016
31. Relationship between phosphorylation of sperm-specific antigen and prognosis of lung adenocarcinoma
- Author
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Hiroyuki Ito, Munetaka Masuda, Yasushi Rino, Fumihiro Oshita, Takashi Oshima, Takuya Nagashima, Yayoi Kimura, Haruhiko Nakayama, Akiko Okayama, Hisashi Hirano, Yohei Miyagi, and Akihide Ryo
- Subjects
Male ,0301 basic medicine ,Oncology ,medicine.medical_specialty ,Lung Neoplasms ,Biophysics ,Adenocarcinoma ,Biochemistry ,Disease-Free Survival ,03 medical and health sciences ,Antigen ,Internal medicine ,medicine ,Humans ,Phosphorylation ,Lung cancer ,Survival rate ,Aged ,Aged, 80 and over ,Lung ,business.industry ,Microfilament Proteins ,Membrane Proteins ,Cancer ,Middle Aged ,medicine.disease ,Neoplasm Proteins ,Survival Rate ,030104 developmental biology ,medicine.anatomical_structure ,Antigens, Surface ,Immunology ,Biomarker (medicine) ,business - Abstract
Lung cancer is generally considered as a highly malignant cancer. A major challenge for the management of lung adenocarcinoma patients is to predict the clinical course of the disease after resection. We analyzed the different levels of phosphorylation of proteins in lung adenocarcinoma tissues between a poor prognosis (PP) group, in which six patients exhibited recurrence within five years after surgery, and a good prognosis (GP) group, in which seven patients did not exhibit recurrence within five years after surgery. We found that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2) [phospho-SSFA2(pS92)] was stimulated in the PP group. Using samples from a total of 46 patients, we investigated the utility of phospho-SSFA2(pS92) to discriminate patients of GP and PP groups, with multiple reaction monitoring (MRM) mass spectrometry. Consequently, we confirmed that the PP group had significantly elevated phospho-SSFA2(pS92) levels. Additionally, no expression of SSFA2 recognized in the normal lung tissues. From these results, we demonstrate that phospho-SSFA2 (pS92) is related to the prognosis of early resected lung adenocarcinomas. Therefore, we suggest that phosphorylation of this protein indicates its role as a potential biomarker and new therapeutic target. Biological significance Lung adenocarcinoma patients often experience a high rate of recurrence after surgery. It is important to discover biomarkers for prognostic prediction and therapeutic targets for treatment of early-stage lung adenocarcinoma. In this study, using tissue samples obtained from patients with lung adenocarcinoma that had been stored for five years at − 80 °C, we identified 13 unique phosphorylated peptides, which were differentially expressed between poor and good prognosis groups. We confirmed that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2)[phospho-SSFA2 (pS92)], was related to poor prognosis. Our study demonstrates that prognostic prediction of early-stage lung adenocarcinoma is possible, and suggests new therapeutic targets for its treatment.
- Published
- 2016
32. Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment
- Author
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Noriaki Arakawa, Hisashi Hirano, Yusuke Masuishi, and Yayoi Kimura
- Subjects
0301 basic medicine ,Signal peptide ,Proteome ,Glycosylphosphatidylinositols ,Biophysics ,GPI-Linked Proteins ,Biochemistry ,GPI anchor binding ,Chromatography, Affinity ,Hydrofluoric Acid ,03 medical and health sciences ,Affinity chromatography ,Cell Line, Tumor ,Humans ,Cell adhesion ,Lipid raft ,Titanium ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,Amino acid ,carbohydrates (lipids) ,030104 developmental biology ,lipids (amino acids, peptides, and proteins) ,Signal transduction - Abstract
Glycosylphosphatidylinositol anchored proteins (GPI-APs) in the outer leaflet of the membrane microdomains, commonly referred to as lipid rafts, play important roles in many biological processes such as signal transduction, cell adhesion, protein trafficking, and antigen presentation. From a topological viewpoint, elucidating the presence and localization of GPI-anchor modification sites (ω-sites) is important for the study of the biophysical properties and anchoring mechanisms of these proteins. However, very few reports have actually identified ω-sites of GPI-APs. To enable large-scale site-specific analysis of GPI anchoring, we developed a method for identification of ω-sites by mass spectrometry by combining titanium dioxide-based affinity purification and hydrogen fluoride treatment. This method was able to identify ~ 3-fold more GPI-APs than our previous method: the new technique identified a total of 73 ω-sites derived from 49 GPI-APs. In 13 of the 49 GPI-APs identified, the GPI-anchor attached to multiple amino acids in the C-terminal site, yielding a variety of different protein species. This method allows us to simultaneously identify many GPI-AP protein species with different ω-sites. We also demonstrated the C-terminal GPI anchor attachment signal peptide, based on information about the GPI anchor binding sites of 49 GPI-APs. Thus, our results provide evidence for new insight into the GPI-anchored proteome and the role of GPI anchoring. Biological significance GPI-anchored proteins (GPI-APs) are localized to the outer leaflet of the plasma membranes. Because the GPI anchor is a complex structure, the identification of GPI-anchored peptides by mass spectrometry has always been considered difficult. To improve the feasibility of large-scale site-specific analysis of GPI anchoring, we developed a method for identification of GPI-anchored peptides by combining titanium dioxide–based affinity purification with hydrogen fluoride treatment. Using this novel technique, we identified a total of 73 ω-sites derived from 49 GPI-APs. These data may help us to develop a comprehensive understanding of the GPI-anchored proteome and the role of GPI anchoring. Moreover, this method could be used to discover GPI-APs as candidate biomarkers.
- Published
- 2016
33. Biological significance of co- and post-translational modifications of the yeast 26S proteasome
- Author
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Hisashi Hirano, Yayoi Kimura, and Ayuko Kimura
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Biophysics ,Protein species ,Biology ,biology.organism_classification ,Biochemistry ,Yeast ,Cell biology ,Mice ,03 medical and health sciences ,030104 developmental biology ,Proteasome ,Biological significance ,Posttranslational modification ,Animals ,Humans ,Phosphorylation ,Protein Processing, Post-Translational ,Function (biology) - Abstract
In yeast (Saccharomyces cerevisiae), co- and post-translational modifications of the 26S proteasome, a large protein complex, were comprehensively detected by proteomic techniques, and their functions were investigated. The presence, number, site, and state of co- and post-translational modifications of the 26S proteasome differ considerably among yeast, human, and mouse. The roles of phosphorylation, Nα-acetylation, Nα-myristoylation, Nα-methylation, and N-terminal truncation in the yeast 26S proteasome were investigated. Although there is only one modification site for either Nα-acetylation, Nα-myristoylation, or Nα-methylation, these modifications play an important role in the functions of the yeast proteasome. In contrast, there are many phosphorylation sites in the yeast 26S proteasome. However, the phosphorylation patterns might be a few, suggesting that tiny modifications exert considerable effects on the function of the proteasome. Biological significance Protein co- and post-translational modifications produce different protein species which often have different functions. The yeast 26S proteasome, a large protein complex, consisting of many subunits has a number of co- and post-translational modification sites. This review describes the effects of the modifications on the function of the protein complex. This article is part of a Special Issue entitled: Protein species. Guest Editors: Peter Jungblut, Hartmut Schluter and Bernd Thiede.
- Published
- 2016
34. ModProt: a database for integrating laboratory and literature data about protein post-translational modifications
- Author
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Hisashi Hirano, Tosifusa Toda, and Yayoi Kimura
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Chemistry ,Proteome ,Posttranslational modification ,Computational biology ,Combinatorial chemistry - Published
- 2016
35. CRMP2 dephosphorylation at S522 rather than hyperphosphorylation as an early-stage marker of Alzheimer's disease
- Author
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Tomoko Akiyama, Ayuko Kimura, Aderemi Caleb Aladeokin, Maho Morishima, Yoshio Goshima, Hisashi Hirano, Shigeo Murayama, Yayoi Kimura, and Takaomi C. Saido
- Subjects
Dephosphorylation ,business.industry ,Applied Mathematics ,General Mathematics ,Cancer research ,Phosphorylation ,Medicine ,Hyperphosphorylation ,Collapsin response mediator protein family ,Disease ,Degeneration (medical) ,Stage (cooking) ,business - Published
- 2020
36. Network-guided analysis of hippocampal proteome identifies novel proteins that colocalize with Aβ in a mice model of early-stage Alzheimer’s disease
- Author
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Hisashi Hirano, Fumio Nakamura, Tomoko Akiyama, Aoi Takahashi-Jitsuki, Ayuko Kimura, Aderemi Caleb Aladeokin, Yoshio Goshima, Yayoi Kimura, Haruko Nakamura, Takashi Saito, Jun Nakabayashi, Daiki Masukawa, Takaomi C. Saido, and Hiroko Makihara
- Subjects
Male ,Proteomics ,0301 basic medicine ,HEPACAM ,Interactome ,Proteome ,Amyloid beta ,Early-stage ,Mice, Transgenic ,Hippocampus ,Glial cell proliferation ,lcsh:RC321-571 ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Alzheimer Disease ,Animals ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Amyloid beta-Peptides ,biology ,Colocalization ,Protein-protein interaction networks ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Neurology ,biology.protein ,Alzheimer’s disease ,030217 neurology & neurosurgery - Abstract
Alzheimer’s disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aβ) plaques accumulation. Numerous pharmacological interventions targeting Aβ plaques accumulation have failed to alleviate AD. Also, the pathological alterations in AD start years before the onset of clinical symptoms. To identify proteins at play during the early stage of AD, we conducted proteomic analysis of the hippocampus of young AppNL-F mice model of AD at the preclinical phase of the disease. This was followed by interactome ranking of the proteome into hubs that were further validated in vivo using immunoblot analysis. We also performed double-immunolabeling of these hub proteins and Aβ to quantify colocalization. Behavioral analysis revealed no significant difference in memory performance between 8-month-old AppNL-F and control mice. The upregulation and downregulation of several proteins were observed in the AppNL-F mice compared to control. These proteins corresponded to pathways and processes related to Aβ clearance, inflammatory-immune response, transport, mitochondrial metabolism, and glial cell proliferation. Interactome analysis revealed several proteins including DLGP5, DDX49, CCDC85A, ADCY6, HEPACAM, HCN3, PPT1 and TNPO1 as essential proteins in the AppNL-F interactome. Validation by immunoblot confirmed the over-expression of these proteins except HCN3 in the early-stage AD mice hippocampus. Immunolabeling revealed a significant increase in ADCY6/Aβ and HEPACAM/Aβ colocalized puncta in AppNL-F mice compared to WT. These data suggest that these proteins may be involved in the early stage of AD. Our work suggests new targets and biomarkers for AD diagnosis and therapeutic intervention.
- Published
- 2019
37. Proteomic analysis of aortic smooth muscle cell secretions reveals an association of myosin heavy chain 11 with abdominal aortic aneurysm
- Author
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Shinichi Suzuki, Motohiko Goda, Shota Yasuda, Keiji Uchida, Masataka Taguri, Ryo Ishiwata, Tomoyuki Minami, Koichi Yoshimura, Yoshihiro Ishikawa, Masataka Matsumoto, Hisashi Hirano, Noriaki Arakawa, Utako Yokoyama, Hitoshi Ogino, Munetaka Masuda, and Nobusato Koizumi
- Subjects
0301 basic medicine ,Male ,Proteomics ,Pathology ,medicine.medical_specialty ,Physiology ,Cell ,Myocytes, Smooth Muscle ,Inflammation ,macromolecular substances ,030204 cardiovascular system & hematology ,Muscle, Smooth, Vascular ,Tissue Culture Techniques ,03 medical and health sciences ,0302 clinical medicine ,Smooth muscle ,Tandem Mass Spectrometry ,Physiology (medical) ,Myosin ,medicine ,Humans ,cardiovascular diseases ,Aorta, Abdominal ,Aged ,Aged, 80 and over ,Myosin Heavy Chains ,business.industry ,Middle Aged ,medicine.disease ,Abdominal aortic aneurysm ,030104 developmental biology ,medicine.anatomical_structure ,Case-Control Studies ,cardiovascular system ,Female ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,Biomarkers ,Aortic Aneurysm, Abdominal ,Chromatography, Liquid - Abstract
Abdominal aortic aneurysm (AAA) is a life-threatening disease, and no disease-specific circulating biomarkers for AAA screening are currently available. We have identified a smooth muscle cell (SMC)-specific biomarker for AAA. We cultured aneurysmal tunica media that were collected from eight patients undergoing elective open-repair surgeries. Secreted proteins in culture medium were subjected to liquid chromatography/tandem mass spectrometry. Myosin heavy chain 11 (myosin-11) was identified as a SMC-specific protein in the tunica media-derived secretions of all patients. We then examined myosin-11 protein concentrations by ELISA in plasma samples from patients with AAA ( n = 35) and age-matched healthy control subjects ( n = 34). Circulating myosin-11 levels were significantly higher in patients with AAA than control subjects. The area under the receiver-operating characteristic curve (AUC) of myosin-11 was 0.77, with a specificity of 65% at a sensitivity of 91%. Multivariate logistic regression analysis showed a significant association between the myosin-11 level and presence of AAA. When the myosin-11 level was combined with hypertension, it improved the prediction of AAA (AUC 0.88) more than hypertension per se. We then investigated the correlation between aortic diameter and circulating myosin-11 levels using AAA serum samples from patients undergoing endovascular aneurysm repair ( n = 20). Circulating myosin-11 levels were significantly correlated with maximum aortic diameter. Furthermore, changes in myosin-11 concentrations from the baseline 12 mo after endovascular aneurysm repair were associated with those in aortic diameter. These data suggest that circulating levels of myosin-11, which is a SMC-specific myosin isoform, may be useful as a biomarker for AAA.NEW & NOTEWORTHY Extensive studies have revealed that inflammation- or proteolysis-related proteins are proposed as biomarkers for abdominal aortic aneurysm (AAA). Changes in these protein concentrations are not specific for smooth muscle, which is a major part of AAA pathologies. Hence, no disease-specific circulating markers for AAA are currently available. We found, using secretome-based proteomic analysis on human AAA tunica media, that myosin heavy chain 11 was associated with AAA. Circulating myosin heavy chain 11 may be a new tissue-specific AAA marker.
- Published
- 2018
38. Increase in constitutively active MEK1 species by introduction of MEK1 mutations identified in cancers
- Author
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Sayaka Ueda, Yayoi Kimura, Tohru Koike, Yoko Ino, Eiji Kinoshita, Emiko Kinoshita-Kikuta, and Hisashi Hirano
- Subjects
0301 basic medicine ,MAP Kinase Signaling System ,Mutant ,Biophysics ,MAP Kinase Kinase 1 ,environment and public health ,Biochemistry ,Analytical Chemistry ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,Neoplasms ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Kinase activity ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Gene ,Protein Kinase Inhibitors ,Chemistry ,Kinase ,Phosphoproteomics ,Phosphoproteins ,Molecular biology ,030104 developmental biology ,HEK293 Cells ,030220 oncology & carcinogenesis ,Mutation ,Mutagenesis, Site-Directed ,Electrophoresis, Polyacrylamide Gel - Abstract
The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.
- Published
- 2018
39. In vitro mouse spermatogenesis with an organ culture method in chemically defined medium
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Yuta Asayama, Tatsuma Yao, Noriaki Arakawa, Hiroyuki Sanjo, Takeru Abe, Takuya Sato, Hiroyuki Yamanaka, Masahiro Yao, Hisashi Hirano, Mitsuru Komeya, Takehiko Ogawa, Kumiko Katagiri, Yoko Ino, and Akio Matsuhisa
- Subjects
0301 basic medicine ,Male ,Physiology ,Retinoic acid ,lcsh:Medicine ,Biochemistry ,chemistry.chemical_compound ,Follicle-stimulating hormone ,Mice ,Reproductive Physiology ,Animal Cells ,Spermatocytes ,Testis ,Medicine and Health Sciences ,Testosterone ,Cell Cycle and Cell Division ,Organ Cultures ,lcsh:Science ,Mice, Inbred ICR ,Multidisciplinary ,Chromosome Biology ,Organic Compounds ,Age Factors ,Serum Albumin, Bovine ,Lipids ,Spermatids ,Meiosis ,Chemistry ,medicine.anatomical_structure ,Cell Processes ,Physical Sciences ,Triiodothyronine ,Biological Cultures ,Cellular Types ,Luteinizing hormone ,Research Article ,Signal Transduction ,Mice, Transgenic ,Tretinoin ,Biology ,In Vitro Techniques ,Organ culture ,Research and Analysis Methods ,Andrology ,03 medical and health sciences ,Organ Culture Techniques ,Albumins ,medicine ,Animals ,Spermatogenesis ,Spermatid ,Ethanol ,lcsh:R ,Organic Chemistry ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Cell Biology ,Luteinizing Hormone ,Sperm ,Spermatogonia ,Culture Media ,Mice, Inbred C57BL ,Chemically defined medium ,030104 developmental biology ,Germ Cells ,chemistry ,Alcohols ,lcsh:Q ,Cattle ,Follicle Stimulating Hormone - Abstract
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.
- Published
- 2018
40. Front Cover: A Phos-tag-based micropipette-tip method for rapid and selective enrichment of phosphopeptides
- Author
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Hisashi Hirano, Emiko Kinoshita-Kikuta, Yayoi Kimura, Maho Kawaguchi, Eiji Kinoshita, Yoko Ino, Tohru Koike, and Elena Tianfei Yuan
- Subjects
Front cover ,Chromatography ,biology ,Chemistry ,Clinical Biochemistry ,Phos ,Pipette ,biology.organism_classification ,Biochemistry ,Analytical Chemistry - Published
- 2017
41. Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells
- Author
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Hisashi Hirano, Kanako Watanabe-Susaki, Yuko Yamanaka, Makoto Asashima, Atsushi Intoh, Hitomi Takada, Hiromu Sugino, Megumi Kowno, Akira Kurisaki, and Kei Enomoto
- Subjects
KOSR ,Pluripotent Stem Cells ,Proteomics ,Cellular differentiation ,Rex1 ,Cell Differentiation ,General Medicine ,differentiation ,Biology ,Stem cell marker ,pluripotency ,Embryonic stem cell ,Leukemia Inhibitory Factor ,ES cells ,General Biochemistry, Genetics and Molecular Biology ,proteins ,Cell biology ,Mice ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Stem cell ,Induced pluripotent stem cell ,Cells, Cultured ,Embryonic Stem Cells ,Adult stem cell - Abstract
Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells.
- Published
- 2015
42. Augmentation of multiple protein kinase activities associated with secondary imatinib resistance in gastrointestinal stromal tumors as revealed by quantitative phosphoproteome analysis
- Author
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Takashi Nagata, Yayoi Kimura, Yuji Sakuma, Yoichi Kurata, Takao Kawakami, Hisashi Hirano, Kayoko Nagata, Yusuke Suzuki, and Yohei Miyagi
- Subjects
Proteomics ,Proteome ,Biophysics ,Antineoplastic Agents ,Biology ,Pharmacology ,Biochemistry ,Piperazines ,Gefitinib ,Cell Line, Tumor ,medicine ,Humans ,Kinase activity ,Protein kinase A ,neoplasms ,Protein kinase B ,Gastrointestinal Neoplasms ,EGFR inhibitors ,GiST ,Imatinib ,Phosphoproteins ,digestive system diseases ,Neoplasm Proteins ,Pyrimidines ,Drug Resistance, Neoplasm ,Benzamides ,Imatinib Mesylate ,Signal transduction ,Protein Kinases ,medicine.drug - Abstract
Mutations in the Kit receptor tyrosine kinase gene ( KIT ), which result in constitutive activation of the protein (KIT), are causally related to the development of gastrointestinal stromal tumors (GISTs). Imatinib, a targeted anticancer drug, exerts a therapeutic effect against GISTs by repressing the kinase activity of KIT. Long-term administration of this drug, however, causes the emergence of imatinib-resistant GISTs. We performed quantitative phosphoproteome analysis using a cell-based GIST model system comprising an imatinib-sensitive GIST cell line (GIST882), GIST882 under treatment with imatinib (GIST882-IM), and secondary imatinib-resistant GIST882 (GIST882-R). Phosphorylated peptides were purified from each cell line using titania-based affinity chromatography or anti-phosphotyrosine immunoprecipitation, and then subjected to LC-MS/MS based quantitative phosphoproteome analysis. Using this method we identified augmentation of the kinase activities of multiple elements of the signal transduction pathway, especially KIT and EGFR. Although, these elements were up-regulated in GIST882-R, no additionally mutated KIT mRNA was found in secondary imatinib-resistant GIST cells. Treatment of GIST882-R with imatinib in combination with gefitinib, an EGFR inhibitor, partially prevented cell growth, implying that EGFR may be involved in acquisition of secondary imatinib resistance in GIST. Biological significance In this study, we performed a quantitative phosphoproteome analysis using a cell culture-based GIST model system. The goal of the study was to investigate the mechanism of acquired resistance in GISTs against imatinib, a molecularly targeted drug that inhibits kinase activity of the KIT protein and that has been approved for the treatment of GISTs. In imatinib-resistant GIST cells, we observed elevated expression of KIT and restoration of its kinase activity, as well as activation of multiple proliferative signaling pathways. Our results indicate that the effects of even so-called ‘molecularly targeted’ drugs, are broad rather than convergent, and that the mechanisms of action of such drugs during continuous administration are extremely complex.
- Published
- 2015
43. Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex
- Author
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Shigeo Ohno, Hisashi Hirano, Mariko Ide, Kazunari Yamashita, Atsushi Suzuki, and Kana T. Furukawa
- Subjects
Ubiquitin-Protein Ligases ,chemical and pharmacologic phenomena ,Cell Count ,Protein Serine-Threonine Kinases ,Cell Line ,Madin Darby Canine Kidney Cells ,DDB1 ,Dogs ,Ubiquitin ,Cell polarity ,Animals ,Drosophila Proteins ,Humans ,Molecular Biology ,Neurofibromin 2 ,biology ,Cell growth ,Tumor Suppressor Proteins ,Cell Cycle ,Cell Polarity ,Cell Biology ,Articles ,G1 Phase Cell Cycle Checkpoints ,Ubiquitin ligase ,Cell biology ,DNA-Binding Proteins ,HEK293 Cells ,biology.protein ,Phosphorylation ,CUL4A ,Carrier Proteins ,G1 phase ,HeLa Cells - Abstract
Lgl is a conserved tumor suppressor suggested to be involved in cell polarity regulation and suppression of cell proliferation. Lgl inhibits formation of the VprBP-DDB1-Cul4A-Roc1 ubiquitin E3 ligase complex, which is implicated in cell cycle progression, by promoting formation of the Lgl-VprBP-DDB1 complex to prevent overproliferation., Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 [VprBP] ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 [VprBP] complex, resulting in G1 arrest.
- Published
- 2015
44. The effects of heat stress on morphological properties and intracellular signaling of denervated and intact soleus muscles in rats
- Author
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Yoichi Kurata, Satoru Takahashi, Akira Higashibata, Susumu Minamisawa, Yoshinobu Ohira, Takashi Ohira, Yayoi Kimura, Yoichiro Kusakari, Hisashi Hirano, Satoshi Furukawa, Masaya Seki, and Takashi Kudo
- Subjects
0301 basic medicine ,Male ,medicine.medical_specialty ,Hot Temperature ,Skeletal Muscle ,Physiology ,Ubiquitin-Protein Ligases ,Muscle Proteins ,Muscular Conditions, Disorders and Treatments ,Thermoregulation ,Signalling Pathways ,Body Temperature ,heat stress ,Tripartite Motif Proteins ,03 medical and health sciences ,Stress, Physiological ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,HSP70 Heat-Shock Proteins ,Rats, Wistar ,Muscle, Skeletal ,Protein kinase B ,Original Research ,Denervation ,neural innervation ,SKP Cullin F-Box Protein Ligases ,biology ,Skeletal muscle ,Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ,Muscle atrophy ,Hsp70 ,Ubiquitin ligase ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Ribosomal protein s6 ,Proteolysis ,biology.protein ,medicine.symptom ,Atrophy ,hypertrophy ,Proto-Oncogene Proteins c-akt ,Intracellular ,Signal Transduction - Abstract
形態: 図版あり, Physical characteristics: Original contains illustrations, Accepted: 2017-06-20, 資料番号: PA1710074000
- Published
- 2017
45. Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1
- Author
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Tomohiro Ishikawa, Hisashi Hirano, Shouichi Higashi, and Yayoi Kimura
- Subjects
0301 basic medicine ,Recombinant Fusion Proteins ,Cell ,Proteinase Inhibitory Proteins, Secretory ,CHO Cells ,Matrix metalloproteinase ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Cricetulus ,Cell Line, Tumor ,Extracellular ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Cell adhesion ,Molecular Biology ,Cell Aggregation ,Serine protease ,biology ,Carcinoma ,Cell Biology ,Cell aggregation ,Peptide Fragments ,Recombinant Proteins ,Cell biology ,Neoplasm Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Amino Acid Substitution ,Solubility ,Tumor progression ,Matrix Metalloproteinase 7 ,Cancer cell ,Colonic Neoplasms ,Mutation ,Proteolysis ,biology.protein ,Enzymology ,RNA Interference ,Cholesterol Esters - Abstract
Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface–bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell–cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141–249, which does not include the serine protease inhibitor domain, has the cell aggregation–inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1–mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7–catalyzed generation of sHAI-1. Considering that MMP-7–induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7–induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies.
- Published
- 2017
46. A Phos-tag-based micropipette-tip method for rapid and selective enrichment of phosphopeptides
- Author
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Elena Tianfei, Yuan, Yoko, Ino, Maho, Kawaguchi, Yayoi, Kimura, Hisashi, Hirano, Emiko, Kinoshita-Kikuta, Eiji, Kinoshita, and Tohru, Koike
- Subjects
Phosphopeptides ,HEK293 Cells ,Pyridines ,Tandem Mass Spectrometry ,Sepharose ,Humans ,Phosphorylation ,Sensitivity and Specificity ,Chromatography, Affinity - Abstract
Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
- Published
- 2017
47. Serum Quantitative Proteomic Analysis Reveals Soluble EGFR To Be a Marker of Insulin Resistance in Male Mice and Humans
- Author
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Tomoko Okuyama, Yasuo Terauchi, Kazuki Tajima, Jun Shirakawa, Hisashi Hirano, Yu Togashi, Ayuko Kimura, and Mayu Kyohara
- Subjects
0301 basic medicine ,Blood Glucose ,Male ,Proteomics ,medicine.medical_specialty ,Proteome ,medicine.medical_treatment ,Blood sugar ,Enzyme-Linked Immunosorbent Assay ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Insulin resistance ,Blood serum ,Internal medicine ,Diabetes mellitus ,medicine ,Animals ,Humans ,Hypoglycemic Agents ,Insulin ,Mice, Knockout ,biology ,business.industry ,Liraglutide ,medicine.disease ,Lipids ,ErbB Receptors ,Mice, Inbred C57BL ,Insulin receptor ,030104 developmental biology ,Diabetes Mellitus, Type 2 ,030220 oncology & carcinogenesis ,biology.protein ,Homeostatic model assessment ,Insulin Resistance ,business ,Biomarkers ,medicine.drug - Abstract
To identify circulating factors as candidates involved in type 2 diabetes mellitus (T2DM), we conducted two different quantitative proteomic analyses: (1) db/db mouse sera were compared with db/+ mouse sera obtained at 4, 8, 12, and 24 weeks of age, and (2) db/db mouse sera from animals treated with liraglutide were compared with sera from animals without liraglutide treatment. Twenty proteins were differentially expressed in db/db mouse sera in the first experiment and eight proteins were differentially expressed in db/db mouse sera after liraglutide treatment in the second experiment. Soluble epidermal growth factor receptor (sEGFR) was identified as a common factor, and its protein level was significantly affected in both experiments. An enzyme-linked immunosorbent assay confirmed that the relatively low serum sEGFR levels in db/db mice were restored by liraglutide treatment. The serum sEGFR levels were elevated in diabetic mice with impaired insulin secretion and decreased in high-fat diet-fed mice and ob/ob mice. The serum sEGFR levels increased after the administration of a dual inhibitor of IGF-1/insulin receptor or streptozotocin. In humans with normal glucose tolerance or T2DM, the serum sEGFR levels were correlated with the fasting blood glucose, fasting serum insulin, homeostatic model assessment of insulin resistance, HbA1c, total cholesterol, low-density lipoprotein cholesterol, and triglycerides levels. These findings suggest that sEGFR might be a biomarker for evaluating insulin resistance or a therapeutic target of liraglutide.
- Published
- 2017
48. 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
- Author
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Ayuko Kimura, Noriaki Arakawa, and Hisashi Hirano
- Subjects
Proteome ,ARID1A ,Immunoblotting ,Molecular Sequence Data ,Down-Regulation ,Biology ,Biochemistry ,Chromatin remodeling ,Ovarian Clear Cell Adenocarcinoma ,Tandem Mass Spectrometry ,Cell Line, Tumor ,medicine ,Humans ,Immunoprecipitation ,Protein phosphorylation ,Amino Acid Sequence ,Phosphorylation ,Clear-cell adenocarcinoma ,Ovarian Neoplasms ,Tumor Suppressor Proteins ,fungi ,DNA Helicases ,Nuclear Proteins ,General Chemistry ,Chromatin Assembly and Disassembly ,medicine.disease ,SWI/SNF ,DNA-Binding Proteins ,Tumor progression ,Cancer research ,Female ,Adenocarcinoma, Clear Cell ,Transcription Factors - Abstract
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.
- Published
- 2014
49. Structural basis of starvation-induced assembly of the autophagy initiation complex
- Author
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Yuko Fujioka, Yayoi Kimura, Nobuo N. Noda, Chika Kondo-Kakuta, Fuyuhiko Inagaki, Yoshinori Ohsumi, Rinji Akada, Hayashi Yamamoto, Sho W. Suzuki, and Hisashi Hirano
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Atg1 ,Molecular Sequence Data ,Autophagy-Related Proteins ,Biology ,Crystallography, X-Ray ,Dephosphorylation ,Sequence Analysis, Protein ,Structural Biology ,Autophagy-initiation complex ,Autophagy ,Serine ,Phosphorylation ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Binding Sites ,Autophagy-related protein 13 ,Yeast ,Protein Structure, Tertiary ,Cell biology ,Transport protein ,Biochemistry ,Carrier Proteins ,Protein Kinases - Abstract
Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established. Here we studied the interactions of yeast Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13 binds tandem microtubule interacting and transport domains in Atg1, using an elongated helix-loop-helix region. Atg13 also binds Atg17, using a short region, thereby bridging Atg1 and Atg17 and leading to Atg1-complex formation. Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17. These observations update the autophagy-initiation model as follows: upon starvation, dephosphorylated Atg13 binds both Atg1 and Atg17, and this promotes PAS assembly and autophagy progression.
- Published
- 2014
50. A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin
- Author
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Kenzaburo Oda, Yuko Ishido, Takeshi Akama, Ryohei Katoh, Hisashi Hirano, Aya Yoshihara, Koichi Suzuki, Mariko Sue, Akiko Okayama, Moyuru Hayashi, Yuqian Luo, and Naoki Hiroi
- Subjects
0301 basic medicine ,endocrine system ,endocrine system diseases ,Immunoprecipitation ,Hormone Replacement Therapy ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Down-Regulation ,Biology ,Thyroglobulin ,Cell Line ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Membrane Microdomains ,Gene expression ,medicine ,Animals ,Protein Isoforms ,RNA, Small Interfering ,Integral membrane protein ,Lipid raft ,Feedback, Physiological ,Thyroid ,beta-Cyclodextrins ,Membrane Proteins ,Endocytosis ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,Gene Expression Regulation ,Microscopy, Fluorescence ,Thyroid Epithelial Cells ,030220 oncology & carcinogenesis ,Cattle ,RNA Interference ,Protein Multimerization ,Hormone - Abstract
Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear.In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells.Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-β-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression.Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.
- Published
- 2016
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