記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Granzyme B (GZMB) is mainly produced by natural killer (NK) cells and activated CD8-positive T cells to induce tumor cell apoptosis. We analyzed the significance of GZMB expression in gastric cancer (GC) tissues from patients with pathological (p)Stage II/III GC after curative resection. PATIENTS AND METHODS: Patients with pStage II/III GC who received curative resection (n=253) were included and the expression levels of GZMB in GC tissues and in the adjacent normal mucosa were measured using quantitative real-time polymerase chain reaction. The expression levels in GC tissues and clinicopathological features and overall survival (OS) were compared in these patients. RESULTS: GZMB expression levels were significantly higher in GC tissues than in the adjacent normal mucosa. GZMB expression levels in GC tissues were not associated with any clinicopathological features. The 5-year OS rate in the high-GZMB expression group was significantly better than that in the low-expression group (5-year survival rate 72.0% vs. 55.7%; p=0.009). Furthermore, on multivariate analysis, high-GZMB expression was an independent factor for better OS (hazard ratio=0.652; 95% confidence interval=0.432-0.987; p=0.043). CONCLUSION: In patients with locally advanced GC after curative resection, GZMB expression in GC tissue may be a useful prognostic marker.

DOI： 10.21873/anticanres.17282

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Membrane-less subcellular compartments play important roles in various cellular functions. Although techniques exist to identify components of cellular bodies, a comprehensive method for analyzing both static and dynamic states has not been established. Here, we apply an antibody-based in situ biotinylation proximity-labeling technique to identify components of static and dynamic nuclear bodies. Using this approach, we comprehensively identify DNA, RNA, and protein components of Cajal bodies (CBs) and then clarify their interactome. By inhibiting transcription, we capture dynamic changes in CBs. Our analysis reveals that nascent small nuclear RNAs (snRNAs) transcribed in CBs contribute to CB formation by assembling RNA-binding proteins, including frontotemporal dementia-related proteins, RNA-binding motif proteins, and heterogeneous nuclear ribonucleoproteins.

DOI： 10.1016/j.celrep.2024.114734

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The organized functioning of the anisotropic myocardial layers-including the inner longitudinal, middle circular, and outer longitudinal layers-is essential for stable systemic circulation. However, the proteomic profile of each myocardial layer has not been studied yet. Here, we aimed to elucidate the layer-specific proteomic profile of human cardiac tissue using microscopic sampling. METHODS: Normal hearts were obtained from five autopsy cases, and cardiomyocytes were microdissected separately from the three myocardial layers of the left ventricle. Histological analysis and shotgun proteomic profiling were performed, followed by immunohistochemical analysis. RESULTS: Histologically, no significant changes were observed among the three layers regarding cardiomyocyte diameter and myocardial fibrosis. Totally 1220 proteins-comprising 9404 peptides-were identified from 15 samples, of which the expression levels of 92 proteins were significantly altered among the layers. Gene ontology enrichment analysis revealed that the proteins specifically elevated in the inner and outer layers mostly belonged to the actin filament-binding protein group. In particular, MYH1 was highly expressed in cardiomyocytes in the outer layer, and CTNNA3 was highly expressed at the intercalated disc in the inner layer. CONCLUSIONS: This is the first report on layer-specific proteomic profiling of human normal hearts. Anisotropic profiles of actin filament-binding proteins in myocardial layers may contribute to the anisotropic contractile and conductive abilities of the heart. Knowledge of the layer-specific proteome profiles of a human heart in the normal state can aid in further research on cardiac pathology, such as the prognosis and treatment of focal myocardial infarction.

DOI： 10.1016/j.prp.2024.155453

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Physical inactivity associated with gravity unloading, such as microgravity during spaceflight and hindlimb unloading (HU), can cause various physiological changes. In this study, we attempted to identify serum proteins whose levels fluctuated in response to gravity unloading. First, we quantitatively assessed changes in the serum proteome profiles of spaceflight mice using mass spectrometry with data-independent acquisition. The serum levels of several proteins involved in the responses to estrogen and glucocorticoid, blood vessel maturation, osteoblast differentiation, and ossification were changed by microgravity exposure. Furthermore, a collective evaluation of serum proteomic data from spaceflight and HU mice identified 30 serum proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, whose levels varied to a similar extent in both gravity unloading models. These changes in serum levels could be involved in the physiological changes induced by gravity unloading. A collective evaluation of serum, femur, and soleus muscle proteome data of spaceflight mice also showed 24 serum proteins, including Igfbp5, Igfbp3, and Postn, whose levels could be associated with biological changes induced by microgravity. This study examined serum proteome profiles in response to gravity unloading, and may help deepen our understanding of microgravity adaptation mechanisms during prolonged spaceflight missions.

DOI： 10.1002/pmic.202300214

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Osteoporosis is characterized by weakened bone microstructure and loss of bone mass. Current diagnostic criteria for osteoporosis are based on the T-score, which is a measure of bone mineral density. However, osteoporotic fragility fractures can occur regardless of the T-score, underscoring the need for additional criteria for the early detection of patients at fracture risk. To identify indicators of reduced bone strength, we performed serum proteomic analysis using data-independent acquisition mass spectrometry with serum samples from two patient groups, one with osteoporosis but no fractures and the other with osteopenia and fragility fractures. Collective evaluation of the results identified six serum proteins that changed to a similar extent in both patient groups compared with controls. Of these, extracellular matrix protein 1 (ECM1), which contributes to bone formation, showed the most significant increase in serum levels in both patient groups. An ELISA-based assay suggested that ECM1 could serve as a serum indicator of the need for therapeutic intervention; however, further prospective studies with a larger sample size are necessary to confirm these results. The present findings may contribute to the provision of early and appropriate therapeutic strategies for patients at risk of osteoporotic fractures. SIGNIFICANCE: This study aimed to identify objective serum indicators of the need for therapeutic intervention in individuals at risk of osteoporotic fracture. Comprehensive proteome analyses of serum collected from patients with osteoporosis but no fractures, patients with osteopenia and fragility fractures, and controls were performed by data-independent acquisition mass spectrometry. Collective evaluation of the proteome analysis data and ELISA-based assays identified serum ECM1 as a potential objective marker of the risk of fragility fractures in patients with osteoporosis or osteopenia. The findings are an important step toward the development of appropriate bone health management methods to improve well-being and maintain quality of life.

DOI： 10.1016/j.jprot.2024.105166

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jcmgh.2024.03.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mitochondrial dysfunction has been implicated in various types of cardiovascular disease including hypertension. Mitochondrial fission fusion balance is critical to mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular disease. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. Here, we have tested the hypothesis that inhibition of mitochondrial fission attenuates the development of hypertension and associated vascular remodeling. In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor, mdivi1, significantly inhibited angiotensin II-induced development of hypertension assessed by radiotelemetry. Histological assessment of hearts and aortas showed that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both the endothelial and medial layers of aortas. Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight-to-body weight ratio as well as by echocardiography. In ex vivo experiments, mdivi1 inhibited vasoconstriction and abolished the enhanced vascular reactivity by angiotensin II in small mesenteric arteries. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature.

DOI： 10.1038/s41440-024-01610-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups, and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. Additionally, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.

DOI： 10.1152/japplphysiol.00300.2023

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The tryptophanyl-tRNA synthetase 1 gene (WARS1), encodes a tryptophan-tRNA synthetase involved in the amino acidification of tryptophan-tRNA and has been reported to be involved in cancer cell growth, metastasis promotion, and drug resistance in a variety of cancers. This study investigated the clinical significance of WARS1 expression as a biomarker in gastric cancer tissues obtained from patients with locally advanced gastric cancer (GC) who underwent radical resection. PATIENTS AND METHODS: WARS1 expression in GC tissues and adjacent normal gastric mucosa of 253 patients with pStage II/III GC who underwent curative resection was determined using quantitative polymerase chain reaction (PCR). Association of WARS1 expression levels, categorized into high and low expression based on the median expression levels, with clinicopathological factors and overall survival (OS) of these patients was assessed. RESULTS: The low-WARS1 expression group had significantly higher serosal invasion, lymph node metastasis, lymphatic invasion, venous invasion, and pathological stage than did the high-WARS1 expression group. OS was significantly worse in the low- than in the high-WARS1 expression group (5-year survival 52.2% vs. 75.9%; p=0.0001). Furthermore, in multivariate analysis, low WARS1 expression was an independent predictor for poor OS (hazard ratio=2.101; 95% confidence interval=1.328-3.322; p=0.002). CONCLUSION: In patients with locally advanced GC, after curative resection, WARS1 expression in GC tissue may be a useful prognostic marker.

DOI： 10.21873/anticanres.16857

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The molecular mechanisms associated with spaceflight-induced biological adaptations that may affect many healthy tissue functions remain poorly understood. In this study, we analyzed temporal changes in the serum proteome of six astronauts during prolonged spaceflight missions using quantitative comprehensive proteome analysis performed with the data-independent acquisition method of mass spectrometry (DIA-MS). All six astronauts participated in a spaceflight mission for approximately 6 months and showed a decreasing trend in T-scores at almost all sites where dual-energy X-ray absorptiometry scans were performed. DIA-MS successfully identified 624 nonredundant proteins in sera and further quantitative analysis for each sampling point provided information on serum protein profiles closely related to several time points before (pre-), during (in-), and after (post-) spaceflight. Changes in serum protein levels between spaceflight and on the ground suggest that abnormalities in bone metabolism are induced in astronauts during spaceflight. Furthermore, changes in the proteomic profile occurring during spaceflight suggest that serum levels of bone metabolism-related proteins, namely ALPL, COL1A1, SPP1, and POSTN, could serve as highly responsive indicators of bone metabolism status in spaceflight missions. This study will allow us to accelerate research to improve our understanding of the molecular mechanisms of biological adaptations associated with prolonged spaceflight.

DOI： 10.1002/pmic.202300328

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The asialoglycoprotein receptor 2 gene (ASGR2) encodes a subunit of the asialoglycoprotein receptor, a transmembrane protein, which has recently been reported to be involved in gastric cancer (GC) progression. This study aimed to investigate the clinical significance of ASGR2 expression in GC tissues of patients with locally advanced gastric cancer (LAGC) after curative resection. PATIENTS AND METHODS: ASGR2 expression was measured in GC tissues and adjacent normal gastric mucosa in 253 patients with pStage II/III GC who underwent curative resection, by using quantitative polymerase chain reaction. We compared the expression levels in GC tissues and adjacent normal stomach mucosa, and evaluated the relationship of its expression in GC tissues with clinicopathological factors and overall survival (OS). RESULTS: ASGR2 expression was significantly associated with lymph node metastasis and venous invasion. The high ASGR2-expression group demonstrated significantly lower survival than the low expression group (5-year survival 55.5% vs. 72.6%; p=0.009). Furthermore, in multivariate analysis, high ASGR2 expression was an independent factor for poor OS (hazard ratio=2.030; 95% confidence interval=1.318-3.127; p=0.001). CONCLUSION: ASGR2 expression in GC tissues may be a useful prognostic marker in patients with LAGC after curative resection.

DOI： 10.21873/anticanres.16824

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Pregnancy zone protein (PZP), encoded by PZP, belongs to the α-2-macroglobulin superfamily, and plays an important role in inflammatory responses and immune cell activation in cancer. However, the relationship between gastric cancer (GC) and PZP is poorly studied. This study investigated the clinical significance of PZP expression in GC tissues of patients with locally advanced GC after curative resection. PATIENTS AND METHODS: Using quantitative polymerase chain reaction, we measured PZP expression in GC tissues and adjacent normal gastric mucosa of 253 patients with pStage II/III GC who underwent curative resection. We compared the expression levels of PZP in GC tissues and adjacent normal gastric mucosa and examined the relationship of PZP expression in GC tissues with clinicopathological factors and overall survival (OS). RESULTS: PZP expression was significantly associated with histology, venous invasion, and pathological stage. The high PZP expression group had significantly worse OS than did the low expression group (5-year survival 48.6% vs. 68.5%, p=0.0003). Furthermore, in multivariate analysis, high PZP expression was an independent factor for poor OS (hazard ratio=1.984, 95% confidence interval=1.307-3.012, p=0.0013). CONCLUSION: In post-curative resection patients with locally advanced GC, PZP expression in GC tissue may be a useful prognostic marker.

DOI： 10.21873/anticanres.16820

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Chitinase-3-like protein 1 (CHI3L1), encoded by CHI3L1, is thought to be involved in growth, invasion, migration, and resistance to chemotherapy in cancer. This study aimed to investigate the clinical significance of CHI3L1 expression as a biomarker in gastric cancer (GC) tissues of patients with locally advanced GC after curative resection. PATIENTS AND METHODS: Quantitative polymerase chain reaction (PCR) was used to determined CHI3L1 expression in GC tissues and adjacent normal gastric mucosa of 253 patients with pStage II/III GC who underwent curative resection. We compared the expression levels in GC tissues and adjacent normal gastric mucosa, and examined the relationship between expression in GC tissues and clinicopathological factors and overall survival (OS) in these patients. RESULTS: CHI3L1 expression was significantly associated with lymph-node metastasis and venous invasion. OS rate was significantly lower in the high- than in the low-CHI3L1 expression group (5-year survival 55.5% vs. 72.6%; p=0.009). Furthermore, in multivariate analysis, high CHI3L1 gene expression was an independent factor for poor OS (hazard ratio=2.030; 95% confidence interval=1.318-3.127; p=0.001). CONCLUSION: In patients with locally advanced GC after curative resection, expression of the CHI3L1 in GC tissue may be a useful prognostic marker.

DOI： 10.21873/anticanres.16813

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Antibody obtained by the coronavirus disease-19 (COVID-19) mRNA vaccine declines over time, and additional vaccinations are offered. It is not clear how repeated vaccination affects humoral immunity in uninfected individuals. We analyzed immunoglobulin G for spike protein (S-IgG) titers in COVID-19 uninfected and infected individuals vaccinated up to six times. The geometric mean S-IgG titers were 575.9 AU/mL and 369.0 AU/mL in those who received 6 and 5 doses less than 180 days after the last vaccination in uninfected subjects. In the 180-360 days after the last vaccination, the geometric mean S-IgG titers were 237.9 AU/mL and 128.6 AU/mL in the uninfected subjects who underwent five-dose and four-dose groups, respectively. Multivariate analysis showed that S-IgG titer increased 1.261-fold with each additional dose of mRNA vaccine. The S-IgG titers were 2.039-fold higher in the COVID-infected subjects compared to uninfected subjects. The positivity rate of nucleocapsid antibodies, suggesting a history of COVID-19, decreased 82% and 30% of COVID-infected cases after 180 and 360 days of infection, respectively. This result suggested that repeated vaccination with the COVID-19 mRNA vaccine may increase antibody titer in uninfected subjects.

DOI： 10.1080/21645515.2023.2278376

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Gravity-dependent physical processes strongly affect the ability of elderly people to maintain musculoskeletal health by reducing muscle atrophy and increasing bone mineral density, thereby increasing quality of life. A need therefore exists to identify molecules in the musculoskeletal system that are responsive to gravitational loading and to establish an objective indicator for the maintenance of healthy musculoskeletal systems. Here, we performed an integrated assessment of the results of soleus muscle proteomic analyses in three model mouse experiments under different gravity environments (hypergravity, hindlimb unloading, and spaceflight). Myl6b, Gpd1, Fbp2, Pvalb, and Actn3 were shown to be gravity-responsive muscle proteins, and alterations in the levels of these proteins indicated changes in muscle fiber type to slow-twitch type due to gravity loading. In addition, immunoblotting and enzyme-linked immunosorbent assays revealed that Pvalb levels in the sera of hindlimb-unloaded mice and osteoporosis patients were higher than in control subjects, suggesting that Pvalb levels might be useful to objectively evaluate soleus muscle atrophy and bone loss.

DOI： 10.1038/s41598-023-42875-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14-Vac8 interaction is constitutive, the Atg38-Atg1 complex interaction and the Vps30-Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis.

DOI： 10.1083/jcb.202210017

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記述言語：英語  

DOI： 10.1016/j.jprot.2023.104976

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer tissues generally have molecular oxygen and serum component deficiencies because of poor vascularization. Recently, we revealed that ICAM1 is strongly activated via lipophagy in ovarian clear cell carcinoma (CCC) cells in response to starvation of long-chain fatty acids and oxygen and confers resistance to apoptosis caused by these harsh conditions. CD69 is a glycoprotein that is synthesized in immune cells and is associated with their activation via cellular signaling pathways. However, the expression and function of CD69 in non-hematological cells is unclear. Here, we report that CD69 is induced in CCC cells as in ICAM1. Mass spectrometry analysis of phosphorylated peptides followed by pathway analysis revealed that CD69 augments CCC cell binding to fibronectin (FN) in association with the phosphorylation of multiple cellular signaling molecules including the focal adhesion pathway. Furthermore, CD69 synthesized in CCC cells could facilitate cell survival because the CD69-FN axis can induce epithelial-mesenchymal transition. Experiments with surgically removed tumor samples revealed that CD69 is predominantly expressed in CCC tumor cells compared with other histological subtypes of epithelial ovarian cancer. Overall, our data suggest that cancer cell-derived CD69 can contribute to CCC progression through FN.

DOI： 10.1111/cas.15774

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記述言語：英語  

DOI： 10.1002/pmic.202200334

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J-GLOBAL

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Primary central nervous system lymphoma (PCNSL) is a malignant neoplasm of the central nervous system that is refractory to treatment and has extremely poor prognosis. One factor hindering the development of therapeutic options for PCNSL is its molecular heterogeneity and the extreme difficulty in establishing in vitro cell lines that permit intensive research on this disease. In the present study, we developed a method to propagate PCNSL cells in vitro using a contacting transwell cell culture system involving brain vascular pericytes. The co-culture system was found to recapitulate the tumor microenvironment that is influenced by the biological activity of adjacent pericytes, and to sustain the survival and proliferation of PCNSL cells in vitro. We further delineated the underlying molecular mechanisms and found that the HGF-c-Met axis may be involved in the long-term in vitro culture of PCNSL cells. Moreover, the peptidylprolyl isomerase Pin1 was found to play a key role in PCNSL cell survival and it sustained proliferation through interactions with key transcription factors related to B-cell lymphomagenesis. These results suggest that our in vitro co-culture system is well suited to analyzing the biological and molecular characteristics of PCNSL, and may contribute to the discovery of new therapeutic interventions.

DOI： 10.3389/fcell.2023.1275519

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: SEC11A gene encodes the SPC18 protein, which has been implicated in tumour progression by inducing the secretion of various growth factors. We investigated the clinical significance of SEC11A expression in gastric cancer (GC) tissues in patients with locally advanced gastric cancer (LAGC) after curative resection. PATIENTS AND METHODS: We estimated SEC11A expression in cancer tissues from 253 pStage II/III GC patients who underwent curative resection using quantitative polymerase chain reaction (PCR) and investigated the relationship of SEC11A expression with clinicopathological factors and survival. RESULTS: SEC11A expression was significantly related to serosal invasion, lymph node metastasis, lymphatic invasion, and pathological stage. The high-SEC11A expression group had a significantly lower survival rate than the low group (5-year survival 52.3% vs. 75.9%; p<0.005). Furthermore, in multivariate analysis, high-SEC11A expression was an independent factor of poor survival (hazard ratio, 2.010; 95% confidence interval=1.303-3.100; p=0.002). CONCLUSION: SEC11A expression in cancer tissue may be a useful prognostic marker in patients with LAGC after curative resection.

DOI： 10.21873/anticanres.16097

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hepatitis B virus (HBV) core antigen (HBc) is a structural protein that forms the viral nucleocapsid and is involved in various steps of the viral replication cycle, but its role in the pathogenesis of HBV infection is still elusive. In this study, we generated a mouse monoclonal antibody (mAb) against HBc and used it in antibody-based in situ biotinylation analysis in order to identify host proteins that interact with HBc. HBc antigen was produced with a wheat germ cell-free protein synthesis system and used to immunize mice. Among the established hybridoma clones, a single clone (mAb #7) was selected and further characterized for its ability in the antibody-based in situ biotinylation analysis to collect host proteins that are in the vicinity of HBc. Using mass spectrometry, we identified 215 HBc-interacting host proteins, three of which bind HBc most significantly under hypoxic conditions. Our results indicate that mAb #7 can be used to systematically identify host proteins that interact with HBc under pathophysiological conditions, and thus may be useful to explore the molecular pathways involved in HBV-induced cytopathogenesis.

DOI： 10.3390/microorganisms10122381

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The COVID-19 pandemic caused by SARS-CoV-2 remains a serious health concern worldwide due to outbreaks of SARS-CoV-2 variants that can escape vaccine-acquired immunity and infect and transmit more efficiently. Therefore, an appropriate testing method for COVID-19 is essential for effective infection control and the prevention of local outbreaks. Compared to reverse-transcription polymerase chain reaction (RT-PCR) tests, antigen tests are used for simple point-of-care testing, enabling the identification of viral infections. In this study, we tested the clinical usefulness of the FUJIFILM COVID-19 Ag test, an antigen test based on silver amplification and immunochromatographic technology. The FUJIFILM COVID-19 Ag test was shown to detect a lower viral concentration as compared to other conventional kits without significant performance loss in detecting prevalent SARS-CoV-2 variants. We tested nasopharyngeal and nasal swabs from a single patient during two different epidemic periods dominated by various SARS-CoV-2 variants. We observed that the sensitivity of the FUJIFILM COVID-19 Ag test was 95.7% and 85.7% in nasopharyngeal and nasal swabs, respectively. These results suggest that the FUJIFILM COVID-19 Ag test is highly sensitive and applicable when RT-PCR testing is unavailable. Furthermore, these results indicate that high-frequency testing using nasal swab specimens may be a valuable screening strategy.

DOI： 10.3390/biomedicines10112801

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記述言語：日本語   出版者・発行元：(一社)日本神経治療学会  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.

DOI： 10.3390/v14102153

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: We sought to identify an optimal combination of survival risk stratification markers in patients with pathological (p) stage II/III gastric cancer (GC) after curative resection. METHODS: We measured the expression levels of 127 genes in pStage II/III GC tissues of two patient cohorts by quantitative polymerase chain reaction (qPCR) and the expression of 1756 proteins between two prognosis (good and poor) groups by proteomic analysis to identify candidate survival stratification markers. Further, immunohistochemistry (IHC) using tumor microarrays (TMAs) in another cohort of patients was performed to identify an optimal biomarker combination for survival stratification in GC patients. RESULTS: secreted protein acidic and rich in cysteine (SPARC), erb-b2 receptor tyrosine kinase 2 (ERBB2), inhibin subunit beta A (INHBA), matrix metallopeptidase-11 (MMP11), tumor protein p53 (TP53), and platelet-derived growth factor receptor-beta (PDGFRB) were identified as candidate biomarkers from qPCR analysis, and SPARC and galectin-10 were obtained from the proteomic analysis. The combination of PDGFRB, INHBA, MMP11, and galectin-10 was identified as the optimal combination of survival risk stratification markers. CONCLUSIONS: A combination of four proteins in GC tissues may serve as useful survival risk stratification markers in patients with pStage II/III GC following curative resection. Our results may facilitate future multicenter prospective clinical trials.

DOI： 10.3390/cancers14184427

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掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription–quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.

DOI： 10.3390/v14071416

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder that affects upper and lower motor neurons; however, its pathomechanism has not been fully elucidated. Using a comprehensive phosphoproteomic approach, we have identified elevated phosphorylation of collapsin response mediator protein 1 (Crmp1) at serine 522 in the lumbar spinal cord of ALS model mice overexpressing a human superoxide dismutase mutant (SOD1G93A). We investigated the effects of Crmp1 phosphorylation and depletion in SOD1G93A mice using Crmp1S522A (Ser522→Ala) knockin (Crmp1ki/ki ) mice in which the S522 phosphorylation site was abolished and Crmp1 knockout (Crmp1 -/-) mice, respectively. Crmp1ki/ki/SOD1G93A mice showed longer latency to fall in a rotarod test while Crmp1-/-/SOD1G93A mice showed shorter latency compared with SOD1G93A mice. Survival was prolonged in Crmp1ki/ki/SOD1G93A mice but not in Crmp1-/-/SOD1G93A mice. In agreement with these phenotypic findings, residual motor neurons and innervated neuromuscular junctions were comparatively well-preserved in Crmp1ki/ki/SOD1G93A mice without affecting microglial and astroglial pathology. Pathway analysis of proteome alterations showed that the sirtuin signaling pathway had opposite effects in Crmp1ki/ki/SOD1G93A and Crmp1-/-/SOD1G93A mice. Our study indicates that modifying CRMP1 phosphorylation is a potential therapeutic strategy for ALS.Significance StatementCollapsin response mediator protein 1 (CRMP1) is an intracellular molecule that mediates semaphorin 3A (Sema3A) signaling. Phosphoproteomic analysis showed that the Semaphorin Neuronal Repulsive Signaling Pathway, which includes Crmp1 phosphorylation at Ser522, is upregulated in SOD1G93A mice that serve as a model of amyotrophic lateral sclerosis (ALS). While deleting both copies of the Crmp1 gene (Crmp1-/- ) leads to deterioration of motor function in SOD1G93A mice, phospho-null Crmp1 (Crmp1ki/ki ) improves motor function while preventing motor neuron loss and denervation of neuromuscular junctions. Among the Sema3A-mediated axon guidance pathways, we propose that CRMP1 phosphorylation is a potential therapeutic target for ALS.

DOI： 10.1523/ENEURO.0133-22.2022

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/pmic.202100216

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pmic.202100216

記述言語：英語  

DOI： 10.1016/j.jprot.2022.104501

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit β1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296-516 aa) or TNPO1 fragment (1-297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296-516-aa fragment of IMB1 and 1-297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

DOI： 10.1016/j.yexcr.2021.112893

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The COVID-19 pandemic is an unprecedented threat to humanity that has provoked global health concerns. Since the etiopathogenesis of this illness is not fully characterized, the prognostic factors enabling treatment decisions have not been well documented. Accurately predicting the progression of the disease would aid in appropriate patient categorization and thus help determine the best treatment option. Here, we have introduced a proteomic approach utilizing data-independent acquisition mass spectrometry (DIA-MS) to identify the serum proteins that are closely associated with COVID-19 prognosis. Twenty-seven proteins were differentially expressed between severely ill COVID-19 patients with an adverse or favorable prognosis. Ingenuity Pathway Analysis revealed that 15 of the 27 proteins might be regulated by cytokine signaling relevant to interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF), and their differential expression was implicated in the systemic inflammatory response and in cardiovascular disorders. We further evaluated practical predictors of the clinical prognosis of severe COVID-19 patients. Subsequent ELISA assays revealed that CHI3L1 and IGFALS may serve as highly sensitive prognostic markers. Our findings can help formulate a diagnostic approach for accurately identifying COVID-19 patients with severe disease and for providing appropriate treatment based on their predicted prognosis.

DOI： 10.1038/s41598-021-98253-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates, e.g., fructo-oligosaccharide (FOS), contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity. We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (μ-g). Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under μ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group). Regardless of FOS ingestion, the absolute wet weights of both muscles tended to decrease, and the fiber phenotype in Sol muscles shifted toward fast-twitch type following μ-g exposure. However, FOS ingestion tended to mitigate the μ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles. These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight.

DOI： 10.1038/s41526-021-00164-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 7 (CCR7) play an important role in the invasion and metastasis of cancer. This study investigated the relationship between relative expression of CXCR4 and CCR7 mRNA, clinicopathological factors, and outcomes in patients with colorectal cancer (CRC). PATIENTS AND METHODS: We studied 202 patients who underwent surgery for CRC. The expression levels of CXCR4 and CCR7 mRNA in cancerous tissue were measured using quantitative real-time reverse-transcriptase polymerase chain reaction. RESULTS: High CCR7 mRNA expression levels in CRC tissues were positively associated with tumour size and were more frequently associated with cancer of the rectum than of the colon. Moreover, outcomes were significantly poorer in patients with high CCR7 mRNA expression than in those with low expression. On multivariate Cox regression analysis, a higher CCR7 mRNA expression level was a significant independent predictor of poorer overall survival in patients with CRC. CONCLUSION: Overexpression of CCR7 mRNA may be a useful independent prognostic factor in patients with CRC.

DOI： 10.21873/anticanres.15259

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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 Ser1452 (<10%) in cell lines derived from ovarian clear cell carcinoma with frequent recurrence and acquired drug resistance. In this study, we tried to elucidate the roles of BRG1 phosphorylation, using cell lines expressing wild-type, phosphorylation-mimic (brg1-S1452D), or non-phosphorylatable (brg1-S1452A) BRG1. Quantitative proteomic analyses revealed upregulation of proteins and phosphoproteins related to linker histone H1s, histone methylation, and protein ubiquitylation in brg1-S1452D cells, which may coordinately promote the chromatin inactivation and ubiquitin-dependent degradation of target proteins. Consistent with these results, brg1-S1452D cells exhibited an increase in condensed chromatin and polyubiquitylated proteins. In brg1-S1452D 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 Ser1452 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.

DOI： 10.1016/j.jprot.2021.104319

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: This study aimed to evaluate the prognostic significance of PLA2G2A expression in patients with locally advanced gastric cancer (GC). PATIENTS AND METHODS: PLA2G2A expression levels in cancerous tissue specimens and adjacent normal mucosa obtained from 134 patients with stage II/III GC who received adjuvant chemotherapy with S-1 after curative resection were measured using real-time quantitative polymerase chain reaction. Subsequently, the associations of PLA2G2A expression with clinicopathological features and survival were evaluated. RESULTS: No association was observed between clinicopathological features and PLA2G2A expression levels. Overall survival was significantly longer in patients with high PLA2G2A expression levels (p=0.022). Multivariate analysis revealed that PLA2G2A expression was a significant, independent prognostic factor (hazard ratio=0.136; 95% confidence interval=0.0185-0.992; p=0.049). CONCLUSION: PLA2G2A mRNA expression may serve as a useful prognostic marker in patients with locally advanced GC who receive curative surgery and adjuvant chemotherapy with S-1.

DOI： 10.21873/anticanres.15146

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Stanniocalcin2 (STC2) is associated with proliferation, invasion, and metastasis in various cancers. We examined the clinical significance of STC2 mRNA expression in patients with colorectal cancer (CRC). PATIENTS AND METHODS: Relative expression levels of STC2 mRNA in CRC tissues and corresponding normal mucosa obtained from 202 patients were measured using quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: Expression of STC2 mRNA was higher in the cancer tissue than in the adjacent normal mucosa. STC2 mRNA expression in cancer tissues was associated with tumour size, liver metastasis, venous invasion, and lymph node metastasis. High expression of STC2 mRNA was significantly associated with poorer postoperative survival (p=0.0003). Multivariate analysis showed that high expression of STC2 mRNA was an independent predictor of postoperative survival. CONCLUSION: High expression of STC2 mRNA in CRC tissue may be a useful prognostic marker in patients with CRC.

DOI： 10.21873/anticanres.14983

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLβ2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLβ2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.

DOI： 10.1096/fj.202002730R

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVE: Kawasaki disease (KD) is a systemic vasculitis in childhood that can lead to coronary artery lesions (CALs). Although early diagnosis and treatment is important for preventing KD patients from development of CALs, diagnosis depends on the clinical features of KD. We studied the usefulness of leucine-rich alpha-2-glycoprotein 1 (LRG1) and angiotensinogen (AGT), previously reported as KD-related proteins, for KD diagnosis and estimation of intravenous immunoglobulin (IVIG) efficacy. METHODS: We undertook a prospective cohort study with patients having two or more KD symptoms in multiple centers in Japan, between July 2017 and February 2019. RESULTS: Two hundred forty-two patients were included. In multivariable analysis, one unit increase in LRG1 was associated with higher odds of KD diagnosis (Odds ratio [OR] 1.02 [95% confidence interval (CI) 1.001-1.03]). Double-positivity for AGT (≥ 26 μg/mL) and LRG1 (≥ 123.5 μg/mL) was an independent biomarker for KD diagnosis in both the total cohort and the subgroup of patients with two to four KD symptoms (OR 5.01 [95% CI 1.86-13.50] and 3.71 [95% CI 1.23-11.16], respectively). There was no association between LRG1/AGT and IVIG efficacy. CONCLUSION: Double-positivity for LRG1 and AGT is an biomarker for KD diagnosis, especially useful in diagnosing incomplete KD from non-KD. Future studies with larger cohorts should seek to determine whether LRG1 and AGT are valuable as definitive data referred at the diagnosis of KD and for estimating the risk of CALs.

DOI： 10.1371/journal.pone.0257138

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic rapidly increases the use of mechanical ventilation (MV). Such cases further require extracorporeal membrane oxygenation (ECMO) and have a high mortality. OBJECTIVE: We aimed to identify prognostic biomarkers pathophysiologically reflecting future deterioration of COVID-19. METHODS: Clinical, laboratory, and outcome data were collected from 102 patients with moderate to severe COVID-19. Interleukin (IL)-6 level and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copy number in plasma were assessed with ELISA kit and quantitative PCR. RESULTS: Twelve patients died or required ECMO owing to acute respiratory distress syndrome despite the use of MV. Among various variables, a ratio of oxygen saturation to fraction of inspired oxygen (SpO2/FiO2), IL-6, and SARS-CoV-2 RNA on admission before intubation were strongly predictive of fatal outcomes after the MV use. Moreover, among these variables, combining SpO2/FiO2, IL-6, and SARS-CoV-2 RNA showed the highest accuracy (area under the curve: 0.934). In patients with low SpO2/FiO2 (< 261), fatal event-rate after the MV use at the 30-day was significantly higher in patients with high IL-6 (> 49 pg/mL) and SARS-CoV-2 RNAaemia (> 1.5 copies/μL) compared to those with high IL-6 or RNAaemia or without high IL-6 and RNAaemia (88% vs. 22% or 8%, log-rank test P = 0.0097 or P < 0.0001, respectively). CONCLUSIONS: Combining SpO2/FiO2 with high IL-6 and SARS-CoV-2 RNAaemia which reflect hyperinflammation and viral overload allows accurately and before intubation identifying COVID-19 patients at high risk for ECMO use or in-hospital death despite the use of MV.

DOI： 10.1371/journal.pone.0256022

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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. 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), and nicotinamide N-methyltransferase (NNMT) are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Thus, 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.

DOI： 10.3390/ijms21217815

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: Glioma-associated oncogene 1 (GLI1) is an important transcription factor in the hedgehog signalling pathway and tumour formation. We evaluated the clinical significance of GLI1 expression as a prognostic factor in patients with locally advanced gastric cancer (GC). PATIENTS AND METHODS: GLI1 expression levels were measured by quantitative real-time polymerase chain reaction analysis of cancerous and adjacent normal mucosa specimens obtained from 142 patients with Stage II/III GC administered adjuvant chemotherapy with S-1 after curative resection. The associations of GLI1 expression with clinicopathological features and survival were evaluated. RESULTS: Clinicopathological features and GLI1 expression showed no association. Overall survival was significantly poorer in the high compared to the low GLI1 expression group (p=0.04). Multivariate analysis revealed that GLI1 expression was a significant independent prognostic factor [p=0.019, hazard ratio (HR)=1.94, 95% confidence interval (CI)=1.70-3.38]. CONCLUSION: GLI1 expression may be a useful prognostic marker in patients with locally advanced GC.

DOI： 10.21873/anticanres.14599

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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."

DOI： 10.1083/jcb.201910063

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited. Mitochondrial dysfunction is frequently associated with mitochondrial fission/fragmentation mediated by the GTPase Drp1 (dynamin-related protein 1). Nuclear factor (NF)-κB, a master regulator of inflammation, is implicated in endothelial dysfunction and resultant complications. Here, we explore a causal relationship between mitochondrial fission and NF-κB activation in endothelial inflammatory responses. In cultured endothelial cells, TNF-α (tumor necrosis factor-α) or lipopolysaccharide induces mitochondrial fragmentation. Inhibition of Drp1 activity or expression suppresses mitochondrial fission, NF-κB activation, vascular cell adhesion molecule-1 induction, and leukocyte adhesion induced by these proinflammatory factors. Moreover, attenuations of inflammatory leukocyte adhesion were observed in Drp1 heterodeficient mice as well as endothelial Drp1 silenced mice. Intriguingly, inhibition of the canonical NF-κB signaling suppresses endothelial mitochondrial fission. Mechanistically, NF-κB p65/RelA seems to mediate inflammatory mitochondrial fission in endothelial cells. In addition, the classical anti-inflammatory drug, salicylate, seems to maintain mitochondrial fission/fusion balance against TNF-α via inhibition of NF-κB. In conclusion, our results suggest a previously unknown mechanism whereby the canonical NF-κB cascade and a mitochondrial fission pathway interdependently regulate endothelial inflammation.

DOI： 10.1161/HYPERTENSIONAHA.120.14686

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. Each cell type can regulate the other's structure and function through a variety of paracrine effectors. Extracellular vesicles (EVs) are released from and transit between cells constituting a novel means of cell-cell communication. Here, we characterized the proteome of EVs released from each vascular cell type and examined the extent to which these vesicles participate in endothelial-vascular smooth muscle cell (VSMC) communication. EVs were collected by ultracentrifugation from media of rat aortic endothelial and smooth muscle cells cultured under serum-free conditions. Vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. Western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial- and smooth muscle-derived EVs as well as proteins unique to each vascular cell type. Functionally, endothelial-derived EVs stimulated vascular cell adhesion molecule-1 (VCAM-1) expression and enhanced leukocyte adhesion in VSMCs while smooth muscle EVs did not elicit similar effects in endothelial cells (ECs). EVs from ECs also induced protein synthesis and senescence in VSMCs. Proteomic analysis of VSMCs following exposure to EC-derived EVs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (HMGB) 1 and HMGB2. Pharmacological blockade HMGB1 and HMGB2 and siRNA depletion of HMGB1 in smooth muscle cells attenuated VCAM-1 expression and leukocyte adhesion induced by EC EVs. These data suggest that EC-derived EVs can enhance signalling pathways which influence smooth muscle cell phenotype.

DOI： 10.1080/20013078.2020.1781427

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記述言語：英語  

Adenoviral vectors are useful tools to manipulate a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR) which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in non-epithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in, transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenovirus with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared to VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of CAR in ECs but not VSMCs. Proteomic analysis as well as mouse aorta immunostaining further suggest significant expression of CAR in ECs but not in VSMCs. In conclusion, adenovirus can effectively transduce the gene of interest in aortic ECs likely due to abundant expression of CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

DOI： 10.1097/FJC.0000000000000821

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

FLCN is a tumor suppressor gene which controls energy homeostasis through regulation of a variety of metabolic pathways including mitochondrial oxidative metabolism and autophagy. Birt-Hogg-Dubé (BHD) syndrome which is driven by germline alteration of the FLCN gene, predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas, pulmonary cysts and less frequently, salivary gland tumors. Here, we report metabolic roles for FLCN in the salivary gland as well as their clinical relevance. Screening of salivary glands of BHD patients using ultrasonography demonstrated increased cyst formation in the salivary gland. Salivary gland tumors that developed in BHD patients exhibited an upregulated mTOR-S6R pathway as well as increased GPNMB expression, which are characteristics of FLCN-deficient cells. Salivary gland-targeted Flcn knockout mice developed cytoplasmic clear cell formation in ductal cells with increased mitochondrial biogenesis, upregulated mTOR-S6K pathway, upregulated TFE3-GPNMB axis and upregulated lipid metabolism. Proteomic and metabolite analysis using LC/MS and GC/MS revealed that Flcn inactivation in salivary gland triggers metabolic reprogramming towards the pentose phosphate pathway which consequently upregulates nucleotide synthesis and redox regulation, further supporting that Flcn controls metabolic homeostasis in salivary gland. These data uncover important roles for FLCN in salivary gland; metabolic reprogramming under FLCN deficiency might increase nucleotide production which may feed FLCN-deficient salivary gland cells to trigger tumor initiation and progression, providing mechanistic insight into salivary gland tumorigenesis as well as a foundation for development of novel therapeutics for salivary gland tumors.

DOI： 10.1016/j.bbrc.2019.11.184

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

DOI： 10.1016/j.jprot.2020.103686

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The pancreatic cancer microenvironment is crucial in cancer development, progression and drug resistance. Cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze the whole cancer-stromal interactome. Here we studied 14 resected pancreatic cancer specimens (8 pancreatic adenocarcinoma (PDAC) patients as a cancer group and 6 intraductal papillary-mucinous adenoma (IPMA) patients as a control). Shotgun proteomics of the stromal lesion dissected with laser captured microdissection (LCM) was performed, and identified 102 differentially expressed proteins in pancreatic cancer stroma. Next, we obtained gene expression data in human pancreatic cancer and normal pancreatic tissue from The Cancer Genome Atlas database (n = 169) and The Genotype-Tissue Expression database (n = 197), and identified 1435 genes, which were differentially expressed in pancreatic cancer cells. To identify relevant and druggable cancer-stromal-interaction targets, we applied these datasets to our in-house ligand-receptor database. Finally, we identified 9 key genes and 8 key cancer-stromal-interaction targets for PDAC patients. Furthermore, we examined FN1 and ITGA3 protein expression in pancreatic cancer tissues using tissue microarrays (TMAs) of 271 PDAC cases, and demonstrated that FN1-ITGA3 had unfavorable prognostic impact for PDAC patients.

DOI： 10.1016/j.canlet.2019.10.031

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

DOI： 10.1016/j.ajhg.2019.11.011

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: The enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1. METHODS: Surgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection. RESULTS: While FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29-5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/low FPGS and low GGH/high FPGS. Patients without adjuvant treatment showed no significant difference. CONCLUSION: GGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.

DOI： 10.1007/s00432-019-03087-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.21873/invivo.11796

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The KIAA1199 gene has been associated with cancer-cell proliferation, but its functions remain poorly studied. Here, we examined the clinical significance of the KIAA1199 mRNA levels in locally advanced gastric cancer (GC). Materials and Methods/Results: Using samples from 254 patients with stage II/III GC, we found significantly higher KIAA1199 levels in cancerous tissues compared to adjacent normal mucosa (ANM). There was no significant relationship between KIAA1199 expression and clinical features. Although overall survival rates (OSR) of patients, who underwent surgery did not correlate with KIAA1199 expression, patients who underwent adjuvant chemotherapy with S-1 and had high KIAA1199 levels displayed significantly lower OSR. KIAA1199 knock down (KIAA1199-KD) suppressed proliferation, invasiveness, and sensitivity of GC cells to 5-fluorouracil (5-FU). CONCLUSION: KIAA1199 expression appears to be a promising prognostic marker in patients with locally advanced GC, who underwent postoperative adjuvant chemotherapy with S-1. KIAA1199 may represent a novel target for GC pharmacotherapy.

DOI： 10.21873/anticanres.13872

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

DOI： 10.1016/j.neures.2019.10.010

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND/AIM: The PRKCI gene encodes Protein kinase C iota. The overexpression of protein kinase C iota is associated with poor outcomes in patients with gastric and other cancers, but the role of the PRKCI gene in gastric cancer is not fully understood. Thus, we evaluated the clinical significance of PRKCI gene expression in gastric cancer. MATERIALS AND METHODS: PRKCI mRNA expression levels in cancerous tissues and adjacent normal mucosa from 398 patients with gastric cancer were measured. Relationships between PRKCI gene expression and clinicopathological characteristics and outcomes were examined. RESULTS: Overall survival was lower in patients with a high expression of PRKCI than in those with low expression (p=0.016). No other relationships were observed. A high PRKCI expression was found to be an independent prognostic factor (p=0.036, HR=1.44, 95%CI=1.02-2.02). CONCLUSION: PRKCI gene expression in cancerous tissue might be a useful prognostic factor in patients with gastric cancer after gastrectomy.

DOI： 10.21873/anticanres.13771

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.nbd.2019.104603

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus-host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response.

DOI： 10.1038/s41467-019-09867-7

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掲載種別：研究論文（学術雑誌）  

DOI： 10.7554/eLife.43088

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その他リンク： https://cdn.elifesciences.org/articles/43088/elife-43088-v2.xml

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

DOI： 10.1016/j.neuint.2018.04.009

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Electrophoresis Society  

DOI： 10.2198/jelectroph.62.11

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier B.V.  

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.

DOI： 10.1016/j.bbapap.2018.05.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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 Gly(451) and Leu(452) 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.

DOI： 10.1074/jbc.M117.796789

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

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-L micropipette tip containing 10L 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 30min 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 25g proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was &gt;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.

DOI： 10.1002/elps.201700175

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Physiological Society  

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α. Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

DOI： 10.14814/phy2.13350

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

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

DOI： 10.1038/srep43732

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Up to 10% of cytosolic proteins are dependent on the mammalian heat shock protein 90 (HSP90) for folding. However, the interactors of its endoplasmic reticulum (ER) paralogue (gp96, Grp94 and HSP90b1) has not been systematically identified. By combining genetic and biochemical approaches, we have comprehensively mapped the interactome of gp96 in macrophages and B cells. A total of 511 proteins were reduced in gp96 knockdown cells, compared to levels observed in wild type cells. By immunoprecipitation, we found that 201 proteins associated with gp96. Gene Ontology analysis indicated that these proteins are involved in metabolism, transport, translation, protein folding, development, localization, response to stress and cellular component biogenesis. While known gp96 clients such as integrins, Toll-like receptors (TLRs) and Wnt co-receptor LRP6, were confirmed, cell surface HSP receptor CD91, TLR4 pathway protein CD180, WDR1, GANAB and CAPZB were identified as potentially novel substrates of gp96. Taken together, our study establishes gp96 as a critical chaperone to integrate innate immunity, Wnt signaling and organ development.

DOI： 10.1371/journal.pone.0169260

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

<p>リン酸化は最も重要なタンパク質の翻訳後修飾であり，ほとんどすべての細胞内プロセスに影響を及ぼす．Phos-tagは，リン酸基に特異的に結合する機能性分子であり，Phos-tagを活用した技術はリン酸化プロテオミクスにおいて強力なツールとなる．Phos-tagアガロースを使用したクロマトグラフィー法は質量分析のための優れたリン酸化ペプチド濃縮法である．Phos-tagアクリルアミドを使用したPhos-tag SDS-PAGEは，タンパク質をリン酸化状態の違いにより分離することができ，リン酸化フォームに関する様々な情報を得ることができる．しかし，現時点ではPhos-tag SDS-PAGEを用いて複数のタンパク質を一度に分離し，リン酸化フォームを解析することは難しい．この点については，技術のさらなる発展を期待するが，Phos-tag SDS-PAGEは，電気泳動を用いたプロテオミクス研究をさらに促進する可能性がある．</p>

DOI： 10.2198/electroph.61.45

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

<p>タンパク質は，翻訳後修飾（PTM）を受けて，その性質を多様に変化させる．そのため，タ ンパク質の機能を理解する上で，質量分析装置を用いた網羅的な解析によって取得できるPTM情報が重要になる．しかし，このような解析によって取得できるタンパク質のPTM情報の大部分は有効活用することができない．そこで，私たちは，PTM情報を統合し，管理するためのシステムを構築し，さらには，タンパク質のPTM情報を集めた独自データベース，ModProt（Post-Translational Modification Map of Proteome）を作成した．今後，ModProtは，PTM研究を推進するための重要なツールとして，また，PTMを標的とした個別化医療実現に向けた強力なツールとして，様々な研究に応用されることが期待される．</p>

DOI： 10.2198/electroph.61.5

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, invivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2(-/-)) mice and examined their behavioral phenotypes. During 24-h home cage monitoring, the activity level during the dark phase of crmp2(-/-) mice was significantly higher than that of wild-type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2(-/-) mice than for WT mice. The duration of social interaction was shorter for crmp2(-/-) mice than for WT mice. Crmp2(-/-) mice also showed mild impaired contextual learning. We then examined the methamphetamine-induced behavioral change of crmp2(-/-) mice. Crmp2(-/-) mice showed increased methamphetamine-induced ambulatory activity and serotonin release. Crmp2(-/-) mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2(-/-) mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

DOI： 10.1111/gtc.12403

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-kappa B signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

DOI： 10.1016/j.immuni.2016.07.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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.

DOI： 10.1016/j.devcel.2016.06.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

DOI： 10.1084/jem.20151088

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.dib.2016.04.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jprot.2016.03.008

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

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 degrees 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. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jprot.2016.03.005

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

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, N-alpha-acetylation, N-alpha-myristoylation, N-alpha-methylation, and N-terminal truncation in the yeast 26S proteasome were investigated. Although there is only one modification site for either N-alpha-acetylation, N-alpha-myristoylation, or N-alpha-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 Bemd Thiede. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jprot.2015.11.016

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FRONTIERS MEDIA SA  

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

DOI： 10.3389/fpls.2016.00031

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記述言語：英語   出版者・発行元：日本電気泳動学会  

Protein post-translational modifications (PTMs) play crucial roles in regulation of protein function and cell signaling, and abnormalities in protein PTMs are both causes and consequences of disease. Mass spectrometry (MS) is widely used to analyze protein PTMs. In this study, we developed an original database, ModProt (Post-Translational Modification Map of Proteome), to integrate our laboratory data and literature information regarding PTM sites. To develop the ModProt database, we constructed a web-based laboratory information management system (LIMS). This system allows us to administer the ModProt database and to view PTM site maps and corresponding protein information including amino acid sequences, official gene symbols, UniProt accessions/IDs, chromosome number/positions, and additional description. The ultimate goal of the ModProt database is to achieve PTM-based diagnosis and personalized medicine through detection of abnormal PTMs by comparing PTM site maps in healthy and disease states using the database.

DOI： 10.2198/jelectroph.60.1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jproteome.5b00082

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Macroautophagy (hereafter referred to as autophagy) degrades various intracellular constituents to regulate a wide range of cellular functions, and is also closely linked to several human diseases(1,2). In selective autophagy, receptor proteins recognize degradation targets and direct their sequestration by double-membrane vesicles called autophagosomes, which transport them into lysosomes or vacuoles(3). Although recent studies have shown that selective autophagy is involved in quality/quantity control of some organelles, including mitochondria and peroxisomes(4), it remains unclear how extensively it contributes to cellular organelle homeostasis. Here we describe selective autophagy of the endoplasmic reticulum (ER) and nucleus in the yeast Saccharomyces cerevisiae. We identify two novel proteins, Atg39 and Atg40, as receptors specific to these pathways. Atg39 localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of part of the nucleus. Atg40 is enriched in the cortical and cytoplasmic ER, and loads these ER subdomains into autophagosomes. Atg39-dependent autophagy of the perinuclear ER/nucleus is required for cell survival under nitrogen-deprivation conditions. Atg40 is probably the functional counterpart of FAM134B, an autophagy receptor for the ER in mammals that has been implicated in sensory neuropathy(5). Our results provide fundamental insight into the pathophysiological roles and mechanisms of 'ER-phagy' and 'nucleophagy' in other organisms.

DOI： 10.1038/nature14506

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain.

DOI： 10.1073/pnas.1421092112

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

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). Phosphoxylated 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. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jprot.2014.12.012

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記述言語：日本語  

DOI： 10.2198/electroph.59.79

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記述言語：日本語  

DOI： 10.2198/electroph.59.126

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle. © 2014, American Society for Microbiology.

DOI： 10.1128/JVI.03170-13

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/nsmb.2822

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

While T-cell responses are directly modulated by Toll-like receptor (TLR) ligands, the mechanism and physiological function of nucleic acids (NAs)-mediated T cell costimulation remains unclear. Here we show that unlike in innate cells, T-cell costimulation is induced even by non-CpG DNA and by self-DNA, which is released from dead cells and complexes with antimicrobial peptides or histones. Such NA complexes are internalized by T cells and induce costimulatory responses independently of known NA sensors, including TLRs, RIG-I-like receptors (RLRs), inflammasomes and STING-dependent cytosolic DNA sensors. Such NA-mediated costimulation crucially induces Th2 differentiation by suppressing T-bet expression, followed by the induction of GATA-3 and Th2 cytokines. These findings unveil the function of NA sensing by T cells to trigger and amplify allergic inflammation.

DOI： 10.1038/ncomms4566

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N-terminal modifications of the yeast Rpt1 subunit, which contains the N-terminal recognition sequence for N-methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N-terminal Pro residue is either unmodified, mono-methylated, or di-methylated, and that this N-methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro-Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N-methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N-methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast. © 2013 WILEY-VCH Verlag GmbH &amp
Co. KGaA, Weinheim.

DOI： 10.1002/pmic.201300207

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

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

DOI： 10.1021/pr4004807

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Growth hormone 1 (GH1), a pituitary hormone, plays a key role in the regulation of growth. Both excess GH1 treatment and overexpression of a GH1 transgene promote growth of salmon, but these animals exhibit physiological abnormalities in viability, fertility and metabolism, which might be related to pituitary function. However, the molecular dynamics induced in the pituitary by excess GH1 remain unknown. In this study, we performed iTRAQ proteome analysis of the amago salmon pituitary, with and without excess GH1 treatment, and found that the expression levels of proteins related to endocrine systems, metabolism, cell growth and proliferation were altered in the GH1-treated pituitary. Specifically, pituitary hormone prolactin (2.29 fold), and somatolactin alpha (0.14 fold) changed significantly. This result was confirmed by proteome and transcriptome analyses of pituitary from the GH1-transgenic (GH1-Tg) amago salmon. The dynamics of protein and gene expression in the pituitary of GH1-Tg amago salmon were similar to those of pituitary treated with excess GH1. Our findings suggest that not only excess GH1 hormone, but also the quantitative changes in other pituitary hormones, might be essential for the abnormal growth of amago salmon. These data will be useful in future attempts to increase the productivity of fish farming. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jprot.2011.12.009

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記述言語：日本語  

DOI： 10.2198/sbk.56.s25

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記述言語：日本語  

DOI： 10.2198/sbk.56.s21

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

N α-Acetyltransferases (NATs) cause the N α-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N α-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N α-acetylated by NatA, and two by NatB. The N α-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N α-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N α-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N α-acetylation is necessary to maintain the ribosome&#039;s protein synthesis function. © 2010 Elsevier B.V.

DOI： 10.1016/j.jprot.2010.12.007

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

"Omics" studies reported to date have dealt with either thoroughly characterized single species or poorly explored meta-microbial communities. However, these techniques are capable of producing highly informative data for the analysis of interactions between two organisms. We examined the bacterial interaction between Escherichia coli O157:H7 (O157) and Bifidobacterium longum (BL) as a pathogenic-commensal bacterial model creating a minimum microbial ecosystem in the gut using dynamic omics approaches, consisting of improved time-lapse 2D-nuclear magnetic resonance (NMR) metabolic profiling, transcriptomic, and proteomic analyses. Our study revealed that the minimum ecosystem was established by bacterial adaptation to the changes in the extracellular environment, primarily by O157, but not by BL. Additionally, the relationship between BL and O157 could be partially regarded as that between a producer and a consumer of nutrients, respectively, especially with regard to serine and aspartate metabolism. Taken together, our profiling system can provide a new insight into the primary metabolic dynamics in microbial ecosystems.

DOI： 10.1021/pr100989c

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

The phosphorylation of heterogeneous nuclear ribonucleoprotein K ( hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.

DOI： 10.1002/pmic.201000349

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

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

DOI： 10.1002/pmic.200900283

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER PHYSIOLOGICAL SOC  

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up-and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.

DOI： 10.1152/physiolgenomics.00095.2007

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Motivation: Although a huge amount of mammalian genomic data does become publicly available, there are still hurdles for biologists to overcome before such data can be fully exploited. One of the challenges for gaining biological insight from genomic data has been the inability to cross-reference transcriptomic and proteomic data using a single informational platform. To address this, we constructed an open-access database that enabled us to cross-reference transcriptomic and proteomic data obtained from immune cells.
Results: The database, named RefDIC (Reference genomics Data-base of Immune Cells), currently contains: (i) quantitative mRNA profiles for human and mouse immune cells/tissues obtained using Affymetrix GeneChip technology; (ii) quantitative protein profiles for mouse immune cells obtained using two-dimensional gel electrophoresis (2-DE) followed by image analysis and mass spectrometry and (iii) various visualization tools to cross-reference the mRNA and protein profiles of immune cells. RefDIC is the first open-access database for immunogenomics and serves as an important information-sharing platform, enabling a focused genomic approach in immunology.

DOI： 10.1093/bioinformatics/btm430

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.

DOI： 10.1002/pmic.200500586

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2004.12.012

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

To identify post-transcriptionally modulated genes at the translational level by phorbol 12-myristate 13-acetate (PMA), we investigated mRNA profiles in the polysomal and the cytoplasmic fractions of U937 cells before and after PMA stimulation using microarrays with 15 017 oligonucleotide probes. Global comparison of the profiles showed that the cytoplasmic distribution of mRNAs was considerably modulated upon PMA stimulation. The results also indicate that PMA post-transcriptionally regulated at least 0.7% of detectable genes in U937 cells. Thus, besides transcriptional modulation by PMA, changes in the translational state of transcripts seem to play a critical role in PMA-induced differentiation of U937 cells. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2004.11.008

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-γ2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

DOI： 10.1073/pnas.0407468101

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

DOI： 10.1002/pmic.200300704

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

N-alpha-Acetylation, catalyzed co-translationally with N-alpha-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3, The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha 1, alpha 2, alpha 3, alpha 4, alpha 7, and beta 3 subunits were acetylated with NAT1, the alpha 5 and alpha 6 subunits were acetylated with MAK3, and the beta 4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha 5 and alpha 6 subunits, respectively, extended the known types of MAK3 substrates, Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the natl mutant or the normal strain were similar in respect to chymotrypsinlike, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N-alpha-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the natl mutant than in the normal strain.

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：(株)医学書院  

＜Point＞●さまざまなプロテオーム解析技術を用いて,患者血清/血漿中蛋白質が解析されている.●COVID-19重症化に伴い,多くの全身性炎症や臓器損傷関連蛋白質が血中で変動している.●COVID-19重症化予測のための新たな血中バイオマーカー候補の同定が期待されている.(著者抄録)

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記述言語：日本語   出版者・発行元：医薬ジャーナル社  

CiNii Books

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その他リンク： http://search.jamas.or.jp/link/ui/2019034319

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2016-4932

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

DOI： 10.2198/electroph.59.79

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

DOI： 10.2198/electroph.59.126

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その他リンク： http://search.jamas.or.jp/link/ui/2018040047

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.100.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2012.0.183.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2012.0.190.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2012.0.139.0

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

Two-dimensional electrophoresis using Phos-tag affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of protein. When we used 2-DE with isoelectric focusing using IPG gel strip in the first dimension and Phos-tag affinity electrophoresis in the second dimension, the nuclear fraction contained more than 20 spots of endogenous heterogeneous nuclear ribonucleoprotein K (hnRNP K) on the 2-DE map. Mass spectrometric analysis indicated that these multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. By applying this two-dimensional electrophoresis map of hnRNP K, we indicated that the multiple forms were differentially modulated in response to external stimulation.<br>

DOI： 10.2198/sbk.56.s21

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.<br>

DOI： 10.2198/sbk.56.s25

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.128.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.121.0

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2010.0.54.1

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：(公社)日本獣医学会  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：(株)メディカルドゥ  

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記述言語：日本語   出版者・発行元：共立出版  

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その他リンク： http://search.jamas.or.jp/link/ui/2004277029

記述言語：日本語   出版者・発行元：(NPO)日本免疫学会  

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE INC  

The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and nat3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0003-9861(02)00639-2

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記述言語：日本語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

The post-translational modification of proteasome has been investigated to determine the relation between function and the post-translational modification in the proteasome. The 26S proteasome is a large protein complex with proteolytic activity, consisting of 20S proteasome and 19S regulatory particle. In yeast, there are at least three N-acetyltransferases (NAT), NAT1, MAK3 and NAT3. Among 14 subunits of the 20S proteasome, the &alpha;1, &alpha;2, &alpha;3, &alpha;4, &alpha;7 and &beta;3 subunits have been found to be N-acetylated with NAT1, the &alpha;5 and &alpha;6 subunits with MAK3, and the &beta;4 subunit with NAT3. It was shown that chymotrypsin-like activity is slightly higher in the NAT1 deletion mutant than the normal strain, suggesting that N-acetylation be related to the proteolytic activity of 20S proteasome. The N-acetylation of 20S proteasome subunits in plants have been studied. In addition to the N-acetylated subunits in yeast, the &beta;6 subunit has been deduced to be N-acetylated in plants. Similarly, N-terminal modification of the 19S regulatory particle has been investigated in yeast. Among 17 subunits identified in the 19S regulatory particle, 13 have been found to be N-terminally modified and at least 11 to be N-acetylated. On the other hand, phosphorylation is well-known post-translational modification in proteins. The phosphorylated subunits have been identified three in the yeast 20S proteasome and two in the mammal 19S regulatory particle. The relation between the proteolytic activity or assembly and phosphorylation of proteasome has been studied, suggesting that the post-translational modification of proteasome is important for its functions.

DOI： 10.2198/sbk.45.129

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記述言語：日本語  

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記述言語：英語   出版者・発行元：日本電気泳動学会  

<i>N</i>-Acetylation, catalyzed with <i>N</i>-acetyltransferase (NAT), is one of the most common co- and post-translational modifications of the &alpha; amino group of eukaryotic proteins. The eukaryotic 20S proteasome contains seven &alpha;-type and seven &beta;-type subunits. We have found that the <i>N</i>-terminus of &alpha;1, &alpha;2, &alpha;3, &alpha;4, &alpha;5, &alpha;6, &alpha;7, &beta;3 and &beta;4 subunits of yeast proteasome is acetylated with NAT. Experiments using the yeast NAT deletion mutant suggested that <i>N</i>-acetylation be related to the proteolytic activity of the 20S proteasome (Kimura et al. J Biol Chem 2000; 275: 4635-9). In the present study, the rice 20S proteasome was purified by ion-exchange chromatography, gel filtration and glycerol density gradient centrifugation from the rice bran, and 14 subunits contained in the 20S proteasome were separated by two-dimensional electrophoresis and the partial amino acid sequence of them was analyzed by Edman degradation. The sequence analysis indicated that among 14 subunits, the &alpha;1, &alpha;2, &alpha;3, &alpha;4, &alpha;5, &alpha;6, &alpha;7, &beta;3 and &beta;4 subunits are <i>N</i>-terminally blocked in rice as well as yeast, and besides these subunits, the &beta;6 subunit is blocked in rice, but not in yeast. This suggests that the &beta;6 subunit in rice may have different functions from that in yeast.

DOI： 10.2198/sbk.44.205

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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開催年月日： 2018年10月

記述言語：英語  

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