Publications by authors named "Jun Adachi"

69 Publications

Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres.

ERJ Open Res 2021 Jan 22;7(1). Epub 2021 Mar 22.

Dept of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

There is an unmet need for novel biomarkers in the diagnosis of multifactorial COPD. We applied next-generation proteomics to serum extracellular vesicles (EVs) to discover novel COPD biomarkers. EVs from 10 patients with COPD and six healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics. Using non-targeted proteomics, we identified 406 proteins, 34 of which were significantly upregulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodelling. We also identified 63 upregulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl-peptidase 2, fibulin-1, and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD. Moreover, six proteins; fibulin-3, tripeptidyl-peptidase 2, UTP-glucose-1-phosphate uridylyl transferase, CD81, CD177, and oncoprotein-induced transcript 3, were correlated with emphysema. Upregulation of fibulin-3 was confirmed by immunoblotting of EVs and immunohistochemistry in lungs. Strikingly, knockout mice spontaneously developed emphysema with age, as evidenced by alveolar enlargement and elastin destruction. We discovered potential pathogenic biomarkers for COPD using next-generation proteomics of EVs. This is a novel strategy for biomarker discovery and precision medicine.
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http://dx.doi.org/10.1183/23120541.00658-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983195PMC
January 2021

ω3 fatty acid metabolite, 12-hydroxyeicosapentaenoic acid, alleviates contact hypersensitivity by downregulation of CXCL1 and CXCL2 gene expression in keratinocytes via retinoid X receptor α.

FASEB J 2021 Apr;35(4):e21354

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan.

ω3 fatty acids show potent bioactivities via conversion into lipid mediators; therefore, metabolism of dietary lipids is a critical determinant in the properties of ω3 fatty acids in the control of allergic inflammatory diseases. However, metabolic progression of ω3 fatty acids in the skin and their roles in the regulation of skin inflammation remains to be clarified. In this study, we found that 12-hydroxyeicosapentaenoic acid (12-HEPE), which is a 12-lipoxygenase metabolite of eicosapentaenoic acid, was the prominent metabolite accumulated in the skin of mice fed ω3 fatty acid-rich linseed oil. Consistently, the gene expression levels of Alox12 and Alox12b, which encode proteins involved in the generation of 12-HEPE, were much higher in the skin than in the other tissues (eg, gut). We also found that the topical application of 12-HEPE inhibited the inflammation associated with contact hypersensitivity by inhibiting neutrophil infiltration into the skin. In human keratinocytes in vitro, 12-HEPE inhibited the expression of two genes encoding neutrophil chemoattractants, CXCL1 and CXCL2, via retinoid X receptor α. Together, the present results demonstrate that the metabolic progression of dietary ω3 fatty acids differs in different organs, and identify 12-HEPE as the dominant ω3 fatty acid metabolite in the skin.
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http://dx.doi.org/10.1096/fj.202001687RDOI Listing
April 2021

Proteomic analysis of urinary and tissue-exudative extracellular vesicles to discover novel bladder cancer biomarkers.

Cancer Sci 2021 Mar 15. Epub 2021 Mar 15.

Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan.

Proteomic analysis of urinary extracellular vesicles (EVs) is a powerful approach to discover potential bladder cancer (BCa) biomarkers, however urine contains numerous EVs derived from the kidney and normal urothelial epithelium, which can obfuscate information related to BCa cell-derived EVs. In this study, we combined proteomic analysis of urinary EVs and tissue-exudative EVs (Te-EVs), which were isolated from culture medium of freshly resected viable BCa tissues. Urinary EVs were isolated from urine samples of 11 individuals (7 BCa patients and 4 healthy individuals), and Te-EVs were isolated from 7 BCa tissues. We performed tandem mass tag (TMT)-labeling liquid chromatography (LC-MS/MS) analysis for both urinary EVs and Te-EVs and identified 1960 proteins in urinary EVs and 1538 proteins in Te-EVs. Most of the proteins identified in Te-EVs were also present in urinary EVs (82.4%), with 55 of these proteins showing upregulated levels in the urine of BCa patients (fold change > 2.0; P < .1). Among them, we selected 22 membrane proteins as BCa biomarker candidates for validation using selected reaction monitoring/multiple reaction monitoring (SRM/MRM) analysis on urine samples from 70 individuals (40 BCa patients and 30 healthy individuals). Six urinary EV proteins (heat-shock protein 90, syndecan-1, myristoylated alanine-rich C-kinase substrate (MARCKS), MARCKS-related protein, tight junction protein ZO-2, and complement decay-accelerating factor) were quantified using SRM/MRM analysis and validated as significantly upregulated in BCa patients (P < .05). In conclusion, the novel strategy that combined proteomic analysis of urinary EVs and Te-EVs enabled selective detection of urinary BCa biomarkers.
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http://dx.doi.org/10.1111/cas.14881DOI Listing
March 2021

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

Genes Environ 2021 Mar 6;43(1). Epub 2021 Mar 6.

Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan.

Background: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).

Results: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay.

Conclusions: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.
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http://dx.doi.org/10.1186/s41021-021-00179-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937321PMC
March 2021

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

Nat Commun 2021 02 24;12(1):1261. Epub 2021 Feb 24.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.
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http://dx.doi.org/10.1038/s41467-021-21396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904790PMC
February 2021

The FAT10 post-translational modification is involved in the lytic replication of Kaposi's sarcoma-associated herpesvirus.

J Virol 2021 Feb 24. Epub 2021 Feb 24.

Department of Cell Biology, Kyoto Pharmaceutical University, Yamashina, Kyoto, Japan

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication, host cell functions including protein expression and post-translational modification pathways are dysregulated by KSHV to promote virus production. Here, we attempted to identify key proteins for KSHV lytic replication by profiling protein expression in the latent and lytic phases using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis, immunoblotting, and quantitative PCR demonstrated that antigen-F (HLA-F) adjacent transcript 10 (FAT10) and UBE1L2 (also known as ubiquitin-like modifier-activating enzyme 6, UBA6) were upregulated during lytic replication. FAT10 is a ubiquitin-like protein (UBL). UBE1L2 is the FAT10-activating enzyme (E1), which is essential for FAT10 modification (FAT10ylation). FAT10ylated proteins were immediately expressed after lytic induction and increased over time during lytic replication. Knockout of UBE1L2 suppressed KSHV production but not KSHV DNA synthesis. In order to isolate FAT10ylated proteins during KSHV lytic replication, we conducted immunoprecipitations using anti-FAT10 antibody and Ni-NTA chromatography of exogenously expressed His-tagged FAT10 from cells undergoing latent or lytic replication. LC-MS/MS was performed to identify FAT10ylated proteins. We identified KSHV ORF59 and ORF61 as FAT10ylation substrates. Our study revealed that the UBE1L2-FAT10 system is upregulated during KSHV lytic replication, and it contributes to viral propagation.Ubiquitin and UBL post-translational modifications, including FAT10, are utilized and dysregulated by viruses for achievement of effective infection and virion production. The UBE1L2-FAT10 system catalyzes FAT10ylation, where one or more FAT10 molecules are covalently linked to a substrate. FAT10ylation is catalyzed by the sequential actions of E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase) enzymes. The E1 enzyme for FAT10ylation is UBE1L2, which activates FAT10 and transfers it to E2/USE1. FAT10ylation regulates the cell cycle, IFN signaling, and protein degradation; however, its primary biological function remains unknown. Here, we revealed that KSHV lytic replication induces UBE1L2 expression and production of FAT10ylated proteins including KSHV lytic proteins. Moreover, UBE1L2 knockout suppressed virus production during the lytic cycle. This is the first report demonstrating the contribution of the UBE1L2-FAT10 system to KSHV lytic replication. Our findings provide insight into the physiological function(s) of novel post-translational modifications in KSHV lytic replication.
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http://dx.doi.org/10.1128/JVI.02194-20DOI Listing
February 2021

Effect of Tricaprin on Cardiac Proteome in a Mouse Model for Triglyceride Deposit Cardiomyovasculopathy.

J Oleo Sci 2020 Nov 12;69(12):1569-1577. Epub 2020 Nov 12.

Laboratory of Cardiovascular Disease, Novel, Non-Invasive, and Nutritional Therapeutics (CNT), Graduate School of Medicine, Osaka University.

Triglyceride deposit cardiomyovasculopathy (TGCV), a rare cardiovascular disorder caused by genetic or acquired dysfunction of adipose triglyceride lipase (ATGL), is marked by defective intracellular lipolysis that results in excessive accumulation of triglycerides (TGs) in the myocardium and coronary arteries, leading to intractable heart failure (HF). We have developed a specific treatment for TGCV using tricaprin, a medium chain TG, as part of a governmental rare disease project in Japan. We recently reported that tricaprin diet improved cardiac TG metabolism and left ventricular function in an ATGL-knockout (KO) mouse, a mouse model for TGCV. Here, we report the effect of tricaprin on the myocardial proteome of KO mice to elucidate the mechanisms of action of tricaprin at protein expression levels. We compared proteomic changes in the hearts of KO mice fed control or tricaprin diet. Tandem mass tag-based shotgun proteomics identified 1832 proteins common to all sample groups. Whole proteomic distribution in the heart was largely up-regulated in KO mice fed control diet. When using cut-off values (>1.5 or <0.67, FDR-adjusted p value<0.01), in fact, 65 proteins were up-regulated whereas only 2 proteins were down-regulated in the hearts of KO mice fed control diet. The former included proteins assigned to "Cardiac Arrhythmia", and "Cardiac Damage" reflecting HF by a toxicity function analysis. One of the latter was Ces1d, which is known to regulate intracellular TG metabolism. These proteomic changes observed in KO mice were dramatically rescued by the tricaprin diet. These results indicated that tricaprin diet ameliorated HF in a TGCV mouse model at protein expression levels and also provided important clues to understand mechanisms for the beneficial effect of tricaprin.
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http://dx.doi.org/10.5650/jos.ess20185DOI Listing
November 2020

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

FASEB J 2020 Dec 15;34(12):16224-16242. Epub 2020 Oct 15.

Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4 ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3 ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4 mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4 mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4 mice. A five-fold increase in the frequency of abnormalities in Kpna4 mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4 mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4 testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.
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http://dx.doi.org/10.1096/fj.202000768RRDOI Listing
December 2020

Vitamin B1 Supports the Differentiation of T Cells through TGF-β Superfamily Production in Thymic Stromal Cells.

iScience 2020 Jul 31;23(9):101426. Epub 2020 Jul 31.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research, and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Asagi Saito, Ibaraki-city, Osaka 567-0085, Japan; Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe-city, Hyogo 650-0017, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita-city, Osaka 565-0871, Japan; International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Graduate School of Medicine and Graduate School of Dentistry, Osaka University, Yamadaoka, Suita-city, Osaka 565-0871, Japan. Electronic address:

Homeostatic generation of T cells, which occurs in the thymus, is controlled at least in part by endogenous cytokines and ligands. In addition, nutritional factors are other key regulators for the homeostasis of host immunity, but whether and how nutrition affects the homeostatic generation of thymocytes remains to be established. Here, we showed that vitamin B1 deficiency resulted in a bias toward the maturation of γδ thymocytes accompanied by decreased differentiation into double-positive thymocytes during thymic involution. These events were mediated through the increased production of TGF-β superfamily members due to the accumulation of branched-chain α-keto acids in thymic stromal cells. These findings revealed essential roles of vitamin B1 in the appropriate differentiation of T cells through the metabolism of thymic stromal cells.
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http://dx.doi.org/10.1016/j.isci.2020.101426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452312PMC
July 2020

Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens.

Theranostics 2020 12;10(5):2115-2129. Epub 2020 Jan 12.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan.

: Cancer phosphoproteomics can provide insights regarding kinases that can be targeted for therapeutic applications. Monitoring the phosphoproteomics in cancer is expected to play a key role in optimizing treatments with kinase inhibitors. Clinical phosphoproteomics in surgical tissues and patient-derived models has been studied intensively. However, the reported data may not accurately reflect the phosphosignaling status in patients due to the effect of ischemia occurring during surgery or changes in the characteristics of cancer cells when establishing the models. In contrast, endoscopic biopsies have an advantage for clinical phosphoproteomics because they can be rapidly cryo-preserved. We aimed to develop a highly sensitive method for phosphoproteomics in endoscopic biopsies of gastric cancer. : Three tumor biopsies and three normal gastric biopsies were obtained by endoscopy at one time, and subjected to our optimized phosphoproteomics. Phosphopeptides were enriched with an immobilized metal affinity chromatography, and labeled with Tandem Mass Tag reagent. Quantified phosphosites were compared between the pairs of tumor/normal biopsies within same patient. Cancer-specific activated pathways and kinases were identified by pathway enrichment analysis and kinase-substrate enrichment analysis. : Our protocol enabled the identification of more than 10,000 class 1 phosphosites from endoscopic biopsies. A comparison between samples from cancer tissue and normal mucosa demonstrated differences in the phosphosignaling, including biomarkers of response to DNA damage. Finally, cancer-specific activation of DNA damage response signaling was validated by additional phosphoproteomics of other patients and western blotting of gastric cancer/normal cells. : In summary, our pioneering approach will facilitate more accurate clinical phosphoproteomics in endoscopic biopsies, which can be applied to monitor the activities of therapeutic kinases and, ultimately, can be a useful tool to precision medicine.
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http://dx.doi.org/10.7150/thno.37623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019165PMC
April 2021

Maternal ω3 docosapentaenoic acid inhibits infant allergic dermatitis through TRAIL-expressing plasmacytoid dendritic cells in mice.

Allergy 2020 08 4;75(8):1939-1955. Epub 2020 Mar 4.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research, and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Ibaraki-city, Japan.

Background: Maternal dietary exposures are considered to influence the development of infant allergies through changes in the composition of breast milk. Cohort studies have shown that ω3 polyunsaturated fatty acids (PUFAs) in breast milk may have a beneficial effect on the preventing of allergies in infants; however, the underlying mechanisms remain to be investigated. We investigated how the maternal intake of dietary ω3 PUFAs affects fatty acid profiles in the breast milk and their pups and reduced the incidence of allergic diseases in the pups.

Methods: Contact hypersensitivity (CHS) induced by 2,4-dinitrofluorobenzene (DNFB) and fluorescein isothiocyanate was applied to the skin in pups reared by mother maintained with diets mainly containing ω3 or ω6 PUFAs. Skin inflammation, immune cell populations, and expression levels of immunomodulatory molecules in pups and/or human cell line were investigated by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. ω3 PUFA metabolites in breast milk and infant's serum were evaluated by lipidomics analysis using LC-MS/MS.

Results: We show that maternal intake of linseed oil, containing abundant ω3 α-linolenic acid, resulted in the increased levels of ω3 docosapentaenoic acid (DPA) and its 14-lipoxygenation products in the breast milk of mouse dams; these metabolites increased the expression of TNF-related apoptosis-inducing ligand (TRAIL) on plasmacytoid dendritic cells (pDCs) in their pups and thus inhibited infant CHS. Indeed, the administration of DPA-derived 14-lipoxygenation products to mouse pups ameliorated their DNFB CHS.

Conclusion: These findings suggest that an inhibitory mechanism in infant skin allergy is induced through maternal metabolism of dietary ω3 PUFAs in mice.
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http://dx.doi.org/10.1111/all.14217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496639PMC
August 2020

The role of actinin-4 (ACTN4) in exosomes as a potential novel therapeutic target in castration-resistant prostate cancer.

Biochem Biophys Res Commun 2020 03 12;523(3):588-594. Epub 2020 Jan 12.

Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Prostate cancer is the second leading cause of cancer death in men in the United States. Several novel therapeutic agents have been developed for castration-resistant prostate cancer (CRPC), but the prognosis for patients with CRPC remains poor. The identification of novel therapeutic targets for CRPC is an urgent issue. Exosomes are small vesicles secreted by a variety of cells, and exosomes derived from cancer cells have been reported to circulate in the patient's bodily fluids, promoting metastasis and invasion. We aimed to identify novel therapeutic targets for CRPC by proteomic analysis of serum exosomes. Exosomes were isolated by ultracentrifugation of sera from 36 men with metastatic prostate cancer: untreated (n = 8), well-controlled with primary androgen deprivation therapy (ADT) (n = 8), and CRPC (n = 20). We identified 823 proteins in the serum exosomes. Six proteins were increased in CRPC patients compared with untreated patients. In contrast, only ACTN4 was increased in the CRPC patients compared to the ADT patients. We focused on ACTN4 as a candidate for targeted therapeutics. ACTN4 was highly expressed in the prostate cancer cell line DU145 as well as exosomes from this line. RNA interference-mediated downregulation of ACTN4 significantly attenuated cell proliferation and invasion in DU145 cells. ACTN4 could be a potential therapeutic target for CRPC.
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http://dx.doi.org/10.1016/j.bbrc.2019.12.084DOI Listing
March 2020

Dietary Omega-3 Fatty Acid Dampens Allergic Rhinitis via Eosinophilic Production of the Anti-Allergic Lipid Mediator 15-Hydroxyeicosapentaenoic Acid in Mice.

Nutrients 2019 11 22;11(12). Epub 2019 Nov 22.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan.

The metabolism and generation of bioactive lipid mediators are key events in the exertion of the beneficial effects of dietary omega-3 fatty acids in the regulation of allergic inflammation. Here, we found that dietary linseed oil, which contains high amounts of alpha-linolenic acid (ALA) dampened allergic rhinitis through eosinophilic production of 15-hydroxyeicosapentaenoic acid (15-HEPE), a metabolite of eicosapentaenoic acid (EPA). Lipidomic analysis revealed that 15-HEPE was particularly accumulated in the nasal passage of linseed oil-fed mice after the development of allergic rhinitis with the increasing number of eosinophils. Indeed, the conversion of EPA to 15-HEPE was mediated by the 15-lipoxygenase activity of eosinophils. Intranasal injection of 15-HEPE dampened allergic symptoms by inhibiting mast cell degranulation, which was mediated by the action of peroxisome proliferator-activated receptor gamma. These findings identify 15-HEPE as a novel EPA-derived, and eosinophil-dependent anti-allergic metabolite, and provide a preventive and therapeutic strategy against allergic rhinitis.
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http://dx.doi.org/10.3390/nu11122868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950470PMC
November 2019

Dietary coconut oil ameliorates skin contact hypersensitivity through mead acid production in mice.

Allergy 2019 08 10;74(8):1522-1532. Epub 2019 Apr 10.

Laboratory of Vaccine Materials, Center for Vaccine and Adjuvant Research and Laboratory of Gut Environmental System, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Ibaraki-city, Osaka, Japan.

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities.
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http://dx.doi.org/10.1111/all.13762DOI Listing
August 2019

S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription.

J Virol 2019 04 3;93(8). Epub 2019 Apr 3.

Department of Virology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4 , and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust. Late (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.
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http://dx.doi.org/10.1128/JVI.01707-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450117PMC
April 2019

The tyrosine kinase v-Src causes mitotic slippage by phosphorylating an inhibitory tyrosine residue of Cdk1.

J Biol Chem 2018 10 22;293(40):15524-15537. Epub 2018 Aug 22.

From the Department of Biochemistry and Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414,

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.
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http://dx.doi.org/10.1074/jbc.RA118.002784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177586PMC
October 2018

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Sci Rep 2018 07 30;8(1):11401. Epub 2018 Jul 30.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan.

Many attempts have been made to reproduce the three-dimensional (3D) cancer behavior. For that purpose, Matrigel, an extracellular matrix from Engelbreth-Holm-Swarm mouse sarcoma cell, is widely used in 3D cancer models such as scaffold-based spheroids and patient-derived organoids. However, severe ion suppression caused by contaminants from Matrigel hampers large-scale phosphoproteomics. In the present study, we successfully performed global phosphoproteomics from Matrigel-embedded spheroids and organoids. Using acetone precipitations of tryptic peptides, we identified more than 20,000 class 1 phosphosites from HCT116 spheroids. Bioinformatic analysis revealed that phosphoproteomic status are significantly affected by the method used for the recovery from the Matrigel, i.e., Dispase or Cell Recovery Solution. Furthermore, we observed the activation of several phosphosignalings only in spheroids and not in adherent cells which are coincident with previous study using 3D culture. Finally, we demonstrated that our protocol enabled us to identify more than 20,000 and nearly 3,000 class 1 phosphosites from 1.4 mg and 150 μg of patient-derived organoid, respectively. Additionally, we were able to quantify phosphosites with high reproducibility (r = 0.93 to 0.95). Our phosphoproteomics protocol is useful for analyzing the phosphosignalings of 3D cancer behavior and would be applied for precision medicine with patient-derived organoids.
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http://dx.doi.org/10.1038/s41598-018-29837-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065387PMC
July 2018

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics.

Synth Syst Biotechnol 2018 Jun 21;3(2):97-104. Epub 2018 Feb 21.

Laboratory for Single Cell Mass Spectrometry, RIKEN Quantitative Biology Center (QBiC), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.

High-sensitivity mass spectrometry approaches using selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems. Both high-sensitivity detection of low-abundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards. Currently, there are various ways for preparing standards, including chemical peptide synthesis, cellular protein expression, and cell-free protein or peptide synthesis. Cell-free protein synthesis (CFPS) or in vitro translation (IVT) systems in particular provide high-throughput and low-cost preparation methods, and various cell types and reconstituted forms are now commercially available. Herein, we review the use of such systems for precise and reliable protein quantification.
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http://dx.doi.org/10.1016/j.synbio.2018.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995455PMC
June 2018

Quantitation of putative colorectal cancer biomarker candidates in serum extracellular vesicles by targeted proteomics.

Sci Rep 2017 10 6;7(1):12782. Epub 2017 Oct 6.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan.

At the moment, there is no sensitive clinical test for detecting early-stage colorectal cancer (CRC). Target proteomics has enabled high-throughput verification of hundreds of biomarker candidate proteins. Using this technology, we verified 725 previously reported CRC biomarker candidate proteins that are functionally correlated with CRC in extracellular vesicles (EVs) from patients. Of these, 356 proteins were quantified, and 34 peptides (22 proteins) showed significant differences in the serum EVs between healthy controls and CRC patients of two independent cohorts (n = 77 and 84). These peptides were evaluated as single or multiple markers, and four single peptides in annexin family proteins and eight combinations of peptides showed area under the curve > 0.9 for discriminating between healthy controls and CRC patients. The sensitivities of annexins A3, A4, and A11 peptides for detecting early-stage CRC greatly exceed those of carcinoembryonic antigen. These peptides are promising biomarkers for early detection of CRC.
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http://dx.doi.org/10.1038/s41598-017-13092-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630664PMC
October 2017

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling.

J Biol Chem 2017 11 15;292(46):18961-18972. Epub 2017 Sep 15.

From the Department of Hepatology,

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN-targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5'-TGA(C/G)TCA), located -218 to -222 bases from the transcription initiation site in the promoter. Of note, in bile duct-ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.
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http://dx.doi.org/10.1074/jbc.M117.793794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706471PMC
November 2017

Deep Phospho- and Phosphotyrosine Proteomics Identified Active Kinases and Phosphorylation Networks in Colorectal Cancer Cell Lines Resistant to Cetuximab.

Sci Rep 2017 09 5;7(1):10463. Epub 2017 Sep 5.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan.

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.
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http://dx.doi.org/10.1038/s41598-017-10478-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585238PMC
September 2017

TKI-addicted ROS1-rearranged cells are destined to survival or death by the intensity of ROS1 kinase activity.

Sci Rep 2017 07 17;7(1):5519. Epub 2017 Jul 17.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.

ROS1 rearrangement is observed in 1-2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation-mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1-expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.
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http://dx.doi.org/10.1038/s41598-017-05736-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514057PMC
July 2017

Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.

J Proteome Res 2017 02 5;16(2):1077-1086. Epub 2016 Dec 5.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki Osaka 567-0085, Japan.

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.
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http://dx.doi.org/10.1021/acs.jproteome.6b00576DOI Listing
February 2017

Phylogenomics and Morphology of Extinct Paleognaths Reveal the Origin and Evolution of the Ratites.

Curr Biol 2017 Jan 15;27(1):68-77. Epub 2016 Dec 15.

School of Life Sciences, Fudan University, SongHu Road 2005, Shanghai 200438, China; The Institute of Statistical Mathematics, Midori-cho 10-3, Tachikawa City, Tokyo 190-8562, Japan; School of Advanced Sciences, SOKENDAI (The Graduate University for Advanced Studies), Hayama-cho, Kanagawa 240-0193, Japan. Electronic address:

The Palaeognathae comprise the flightless ratites and the volant tinamous, and together with the Neognathae constitute the extant members of class Aves. It is commonly believed that Palaeognathae originated in Gondwana since most of the living species are found in the Southern Hemisphere [1-3]. However, this hypothesis has been questioned because the fossil paleognaths are mostly from the Northern Hemisphere in their earliest time (Paleocene) and possessed many putative ancestral characters [4]. Uncertainties regarding the origin and evolution of Palaeognathae stem from the difficulty in estimating their divergence times [1, 2] and their remarkable morphological convergence. Here, we recovered nuclear genome fragments from extinct elephant birds, which enabled us to reconstruct a reliable phylogenomic time tree for the Palaeognathae. Based on the tree, we identified homoplasies in morphological traits of paleognaths and reconstructed their morphology-based phylogeny including fossil species without molecular data. In contrast to the prevailing theories, the fossil paleognaths from the Northern Hemisphere were placed as the basal lineages. Combined with our stable divergence time estimates that enabled a valid argument regarding the correlation with geological events, we propose a new evolutionary scenario that contradicts the traditional view. The ancestral Palaeognathae were volant, as estimated from their molecular evolutionary rates, and originated during the Late Cretaceous in the Northern Hemisphere. They migrated to the Southern Hemisphere and speciated explosively around the Cretaceous-Paleogene boundary. They then extended their distribution to the Gondwana-derived landmasses, such as New Zealand and Madagascar, by overseas dispersal. Gigantism subsequently occurred independently on each landmass.
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http://dx.doi.org/10.1016/j.cub.2016.10.029DOI Listing
January 2017

Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines.

Sci Rep 2016 11 11;6:36946. Epub 2016 Nov 11.

Laboratory of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, N-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.
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http://dx.doi.org/10.1038/srep36946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105139PMC
November 2016

Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON.

Sci Rep 2016 Sep 29;6:34472. Epub 2016 Sep 29.

Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan.

In some fibroblasts, casein kinase 1α (CK1α) is localized to nuclear speckles, which are sub-nuclear compartments supplying splicing factors, whereas it is recruited on keratin filaments in colorectal cancer cells such as DLD1 cells. In order to obtain a deeper understanding of why CK1α is localized to these different subcellular sites, we herein elucidated the mechanisms underlying its localization to nuclear speckles. CK1α and FAM83H were localized to nuclear speckles in RKO and WiDr colorectal cancer cells, which do not express simple epithelial keratins, and in DLD1 cells transfected with siRNAs for type I keratins. The localization of FAM83H to nuclear speckles was also detected in colorectal cancer cells with a poorly organized keratin cytoskeleton in colorectal cancer tissues. Using an interactome analysis of FAM83H, we identified SON, a protein present in nuclear speckles, as a scaffold protein to which FAM83H recruits CK1α. This result was supported by the knockdown of FAM83H or SON delocalizing CK1α from nuclear speckles. We also found that CK1δ and ε are localized to nuclear speckles in a FAM83H-dependent manner. These results suggest that CK1 is recruited to nuclear speckles by FAM83H and SON in the absence of an intact keratin cytoskeleton.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041083PMC
http://dx.doi.org/10.1038/srep34472DOI Listing
September 2016

Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient.

Anal Chem 2016 08 2;88(16):7899-903. Epub 2016 Aug 2.

Laboratory of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition , Ibaraki, Osaka 567-0085, Japan.

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques.
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http://dx.doi.org/10.1021/acs.analchem.6b01232DOI Listing
August 2016

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes.

Sci Rep 2016 05 25;6:26557. Epub 2016 May 25.

Department of Biochemistry &Molecular Biology, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan.

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.
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http://dx.doi.org/10.1038/srep26557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879633PMC
May 2016