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OBJECT: Functions of layilin, a type 1 transmembrane protein with a C-type lectin motif, remain to be clarified. We here investigated precise intracellular localization of layilin and the location-related functions. METHODS: We used HEK293T cells to assess the co-localization of layilin with different individual organelle markers by double immunostaining. We then investigated mitochondrial morphology in layilin-knockdown (KD) conditions, also with immunostaining. Next, we measured amounts of proteins involved in regulation of mitochondrial dynamics, DRP1, pS616-DRP1, mitofusin1, mitofusin2, CDK1, pY15-CDK1, and cyclin B1, in layilin-KD cells versus control cells by Western blot. Furthermore, by using layilin-knockout (KO) cells, amounts of CDK1 and pY15-CDK1 as well as mitochondrial morphology were investigated. RESULT: We found that layilin localized to mitochondria rather than the other organelles. Small round-shape mitochondria were observed in control cells, whereas elongated and highly connected mitochondria were observed in layilin-KD cells. Amounts of active DRP1 (pS616-DRP1) and total DRP1 were significantly smaller in layilin-KD cells than in controls. Amounts of inactive CDK1 (pY15-CDK1) were significantly larger in layilin-KD cells than in controls. No other tested molecules were significantly altered in layilin-KD cells. Amounts of inactive CDK1 were significantly larger in layilin-KO cells than in wild type (WT) cells. Small round-shape mitochondria were observed in WT cells, whereas elongated and highly connected mitochondria were observed in layilin-KO cells. CONCLUSION: We here demonstrated that layilin played a role in the maintenance of fragmented mitochondria in mitochondrial dynamics and that this function needed CDK1 and DRP1 activation. Our data unveiled a novel function for layilin, regulation of mitochondrial dynamics.
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Proteína Quinasa CDC2/metabolismo , Dinaminas/metabolismo , Lectinas Tipo C/metabolismo , Dinámicas Mitocondriales , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mitocondrias/metabolismo , Modelos BiológicosRESUMEN
A disintegrin and metalloprotease 17 (ADAM17) catalyzes the cleavage and release of the ectodomains of its substrates at the cell surface in a process termed ectodomain shedding. However, not all ADAM17 substrates have been identified. Here, we used cell surface protein-specific labeling and proteomic approaches to detect and identify ADAM17 substrates. HeLa cell surface proteins were labeled with a fluorescent dye and cultured with or without TAPI-2, an ADAM17 inhibitor. Labeled proteins released into the culture medium were detected by 2-dimensional gel electrophoresis (2DE). Protein spots showing decreased intensity in response to TAPI-2 were selected as substrates of ADAM17 or their binding proteins, and identified by mass spectrometry. ADAM17 knockdown was preformed to examine the behavior of identified proteins. Of 347 proteins detected by 2DE, 49 showed lower intensity in TAPI-2 (+) than in TAPI-2 (-) samples (p < 0.05), and were considered as candidate substrates of ADAM17. Mass spectrometric analysis of 14 protein spots showing >50% decreased intensity identified clusterin as a novel ADAM17 substrate, in addition to known substrates such as desmoglein-2. Western blot analysis showed that ADAM17 knockdown decreased the levels of clusterin fragments cleaved and released from the cell surface. The results identified clusterin as a novel ADAM17 substrate. The method used to identify clusterin could be used to identify the substrates of other sheddases involved in ectodomain shedding.
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BACKGROUND: Malignant gliomas are characterized by high invasive ability. In this study, we investigated roles of layilin, a C-type lectin-homologous protein, in the invasive ability of malignant glioma cells. METHODS: Expression of layilin was investigated by western blotting in the malignant glioma cell lines of U251-MG, A172, and T98G and in astrocytes. The effects of layilin-knockdown on the expression and protein levels of snail family transcriptional repressor 1 (SNAI1), a transcriptional factor involved in the acquisition and enhancement of invasive ability in malignant gliomas, and on the expression of its target genes, matrix metalloproteinase 2 (MMP2), MMP9, and collagen type I alpha 1 chain (COL1A1), were investigated by qPCR and/or western blotting. Furthermore, the effects of layilin-knockdown on the expression and protein levels of metastasis associated 1 family member 3 (MTA3), a transcriptional repressor of SNAI1, were also investigated by qPCR and western blotting. Finally, the effects of layilin-knockdown on the invasive ability of the cells were investigated by a wound healing assay. RESULTS: All the tested malignant glioma cells highly expressed layilin, compared to astrocytes, one of representative glial cell types. Layilin-knockdown reduced SNAI1 both at the mRNA and protein levels in A172 cells, and consequently mRNA levels of MMP2, MMP9, and COL1A1 were also reduced. Furthermore, layilin-knockdown increased nuclear protein levels of MTA3 in A172 cells. Notably, layilin-knockdown suppressed the invasive ability of the cells. CONCLUSION: Layilin up-regulates the expression of SNAI1 via down-regulation of MTA3. This process enhances the invasive ability of malignant glioma cells.
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Glioma/metabolismo , Lectinas Tipo C/metabolismo , Invasividad Neoplásica/fisiopatología , Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Regulación de la Expresión Génica/genética , Glioma/fisiopatología , Humanos , Lectinas Tipo C/fisiología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción de la Familia Snail/fisiología , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. RESULTS: By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (pâ¯<â¯0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. CONCLUSION: We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.
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Proteínas de la Membrana/efectos de los fármacos , Sarcoma Sinovial/metabolismo , Sulfasalazina/farmacología , Biotinilación/métodos , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Mesalamina/farmacología , Sarcoma Sinovial/patología , Sulfapiridina/farmacología , Sulfasalazina/metabolismo , Electroforesis Bidimensional Diferencial en Gel/métodosRESUMEN
BACKGROUND: To understand the roles of serum exosomes in rheumatoid arthritis (RA), we comprehensively investigated the protein profiles of serum exosomes in patients with RA. METHODS: Exosomes were isolated from serum samples obtained from 33 patients (12 with active RA [aRA], 11 with inactive RA [iRA], 10 with osteoarthritis [OA]) and 10 healthy donors (HLs). Proteins extracted from the exosomes were separated by two-dimensional differential gel electrophoresis (2D-DIGE) and identified by mass spectrometry. RESULTS: In total, 204 protein spots were detected by 2D-DIGE. In the aRA, iRA, and OA groups, 24, 5, and 7 spots showed approximately ≥ ±1.3-fold intensity differences compared with the HL group, respectively. We were able to identify proteins in six protein spots. Among them, the protein spot identified as Toll-like receptor 3 (TLR3) showed approximately 6-fold higher intensity in the aRA group than in the other groups. CONCLUSIONS: Patients with active RA possessed considerably different protein profiles of serum exosomes from patients with iRA, patients with OA, and healthy donors. The unique protein profile of serum exosomes, such as the possession of abundant TLR3 fragments, may reflect the pathophysiology of active RA.
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BACKGROUND: Psoriasis is a refractory inflammatory disease, however, its pathophysiology is still not fully understood. OBJECTIVE: We tried to identify novel serum peptides associated with the pathophysiology of psoriasis. METHODS: Serum peptides from 24 patients with psoriasis vulgaris (PV), 10 patients with psoriatic arthritis (PsA), 14 patients with atopic dermatitis (AD), and 23 healthy control (HC) subjects were analyzed by mass spectrometry. The effects of some peptides on the secretion of humoral factors from dermal cells were investigated by cytokine arrays and ELISAs. RESULTS: A total of 93 peptides were detected. 24, 20, 23, and 2 peptides showed at least 1.2-fold difference in ion intensity between the psoriasis (PV+PsA) and HC groups, between the PV+PsA and AD groups, between the PV and PsA groups, and between patients with severe-to-moderate PV (n=6) and those with mild PV (n=18), respectively (p<0.05). 13 out of 27 peptides that showed at least 1.5-fold ion intensity difference in the abovementioned 4 comparisons were identified. The parent proteins of the identified peptides included a coagulation factor, proteins involved in the maintenance of skin, and a protein relating to cytoskeleton. We focused on 2 peptides that were increased in the PV+PsA group: a fibrinogen α chain-derived peptide (1462m/z), the unmodified form of which was fibrinopeptide A-des-alanine (FPAdA), and a filaggrin (FLG)-derived peptide (1977m/z), a modified form of FLG2099-2118 (Q2099pE, Q2115E; FLG-pEE). FPAdA stimulation increased the secretion of GROα from dermal microvascular endothelial cells (dMVECs) and decreased the secretion of lipocalin-2 from keratinocytes in comparison to FPAdA-resequenced peptide stimulation (GROα, 280.9±7.3pg/mL vs. 229.6±5.0pg/mL, p<0.001; lipocalin-2, 273±13pg/mL vs. 350±10pg/mL, p<0.01). Interestingly, FLG-pEE stimulation decreased the secretion of GROα, IL-8, and MCP-1 from dMVECs in comparison to FLG-derived control peptide stimulation (GROα, 844.3±47.5pg/mL vs. 1038.5±96.9pg/mL, p<0.05; IL-8, 2240.1±172.6pg/mL vs. 3221.8±523.7pg/mL, p<0.05; MCP-1, 4057.8±157.2pg/mL vs. 4619.1±213.4pg/mL, p<0.05). CONCLUSIONS: The results suggested that some serum peptides are involved in the pathophysiology of psoriasis, regulating the secretion of inflammatory chemokines and an antimicrobial protein. The modulation of serum peptides may be a potential therapeutic strategy for psoriasis.
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Proteínas Sanguíneas/fisiología , Inflamación/etiología , Péptidos/sangre , Psoriasis/etiología , Adulto , Anciano , Proteínas Sanguíneas/análisis , Femenino , Fibrinopéptido A/fisiología , Proteínas Filagrina , Humanos , Proteínas de Filamentos Intermediarios/fisiología , Masculino , Persona de Mediana Edad , Psoriasis/sangre , Psoriasis/terapiaRESUMEN
BACKGROUND: In universal hepatitis B (HB) vaccination, single vaccine-derived polyclonal anti-HBs antibodies (anti-HBs) need to inhibit infection of HB viruses (HBV) of non-vaccine genotypes. We experimentally addressed this issue. METHODS: Anti-HBs-positive sera were obtained by vaccination with genotype A- or C-derived HBs antigen (HBsAg, gtA-sera or gtC-sera). Their reactivity to genotype A- and C-derived HBsAg (gtA-Ag and gtC-Ag) was measured by ELISA. The capacity of sera to neutralize HBV was evaluated using an in vitro infection model. RESULTS: Of 135 anti-gtA-Ag-reactive gtA-sera, 134 (99.3%) were anti-gtC-Ag-reactive. All (100%) 120 anti-gtC-Ag-reactive gtC-sera were anti-gtA-Ag-reactive. The reactivity to gtA-Ag was strongly correlated with that to gtC-Ag (gtA-sera, ρ = 0.989; gtC-sera, ρ = 0.953; p < 0.01). In gtA-sera (n = 10), anti-HBs to gtA-Ag were less completely absorbed with gtC-Ag (96.4%) than with gtA-Ag (100%, p < 0.05). Similarly, in gtC-sera (n = 10), anti-HBs to gtC-Ag were less completely absorbed with gtA-Ag (96.0%) than with gtC-Ag (100%, p < 0.01). Thus, 3.6 and 4.0% of anti-HBs in gtA-sera and gtC-sera were vaccine genotype HBsAg-specific, respectively. In the neutralization test, gtA-sera (n = 4) and gtC-sera (n = 3) with anti-HBs titers adjusted to 100 mIU/mL equally inhibited genotype C HBV infection (92.8 vs. 95.4%, p = 0.44). However, at 30 mIU/mL, the gtA-sera less effectively inhibited infection than the gtC-sera (60.2 vs. 90.2%, p < 0.05). CONCLUSIONS: Vaccination with genotype A- or C-derived HBsAg provided polyclonal anti-HBs that sufficiently bound to non-vaccine genotype HBsAg. However, a small portion of anti-HBs were specific to the vaccine genotype HBsAg. High anti-HBs titers would be required to prevent HBV infection of non-vaccine genotypes. UMIN/CTR UMIN000014363.
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Anticuerpos contra la Hepatitis B/sangre , Vacunas contra Hepatitis B/administración & dosificación , Virus de la Hepatitis B/genética , Hepatitis B/prevención & control , Adolescente , Adulto , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Humanos , Masculino , Vacunación , Adulto JovenRESUMEN
BACKGROUND: Recently, it has been reported that hypoxia highly enhances expression of peptidylarginine deiminase (PAD) 4 and production of citrullinated proteins in some tumor cells. However, little is known about malignant gliomas on this issue. Therefore, we here investigated whether expression of PADs was induced by hypoxia and whether PADs citrullinated intracellular proteins if induced using U-251 MG cells of a human malignant glioma cell line. METHODS: Expression of PADs in U-251 MG cells, cultured under hypoxia or normoxia for 24 h, was investigated by quantitative polymerase chain reaction (qPCR). Citrullination of proteins in the cells and the cell lysates incubated for 48 h with or without Ca2+ was detected by western blotting. Citrullinated proteins were identified by mass spectrometry. RESULTS: The mRNA levels of PAD1, 2, 3, and 4 were up-regulated by hypoxia in a hypoxia-inducible factor-1-dependent manner in U-251 MG cells. In spite of the increased expression, intracellular proteins were not citrullinated. However, the induced PADs citrullinated U-251 MG cell-derived proteins when the cells were lysed. Multiple proteins citrullinated by hypoxia-induced PADs were identified. In addition, the extracellular domain of vascular endothelial growth factor receptor 2 was citrullinated by human PAD2 in vitro. CONCLUSION: Our data may contribute to understanding of pathophysiology of malignant gliomas from the aspects of protein citrullination.
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Citrulina/metabolismo , Glioma/metabolismo , Hidrolasas/metabolismo , Oxígeno/metabolismo , Hipoxia Tumoral , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina ProteicaRESUMEN
Nuclear factor-κB (NF-κB) is the most potent proinflammatory transactivator, and an inhibitor of NF-κB is a good antiinflammatory drug. Glucocorticoids (GCs) are the strongest and the most frequently used antiinflammatory drugs. GC-bound glucocorticoid receptor (GR) inhibits the transcriptional activity of NF-κB and thereby suppresses a broad range of inflammatory processes. Concurrently, in whole body outside the inflammation area, the GR exerts a lot of hormone action, which results in severe side effects. There is a long-awaited need for a new NF-κB inhibitor. Previously we found a small nuclear acidic protein (named MTI-II, also known as Zn2+-binding protein or parathymosin), which worked as a coactivator of GR. Here we showed that overexpression of MTI-II inhibited a transcriptional activity of NF-κB independently of GCs and the GR. Vise versa, RNA interference suppression of inherent MTI-II enhanced the transcriptional activity of NF-κB. An immunoprecipitation analysis showed that MTI-II precipitated NF-κB after the stimulation of TNFα. Deletion mutants of MTI-II showed that central acidic region is essential for the inhibition of the transcriptional activity of NF-κB. These results suggest that MTI-II would be an inherent inhibitor that interacts with NF-κB. Next, we constructed MTI-II-based antiinflammatory drugs (three fusion proteins of MTI-II with a protein transduction domain and a fusion peptide of the central acidic region with protein transduction domain). These drugs had significant antiinflammatory effects on acute inflammation models and on animal models of human chronic inflammation diseases without an increase of the blood glucose level and repeated-dose toxicity. The MTI-II-based antiinflammatory drug will be a good alternative of GCs.
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Antiinflamatorios/farmacología , FN-kappa B/genética , Receptores de Glucocorticoides/metabolismo , Timosina/análogos & derivados , Activación Transcripcional/efectos de los fármacos , Animales , Células COS , Chlorocebus aethiops , Diseño de Fármacos , Células HeLa , Humanos , FN-kappa B/metabolismo , Unión Proteica , Timosina/metabolismo , Transactivadores/metabolismoRESUMEN
To identify proteins related to the pathophysiology of aortic valve stenosis (AS), we investigated the protein profiles of AS aortic valves. Specifically, proteins were extracted from a thickened and calcified area (AS-C) and an apparently non-thickened and non-calcified area (AS-N) in an identical aortic valve leaflet in each of 6 AS patients. The proteins were then separated by 2-dimensional gel electrophoresis (2DE). Protein spots detected by 2DE were compared between the AS-C and AS-N samples. Protein spots of interest were subjected to protein identification by mass spectrometry.In total, 670 protein spots were detected by 2DE, 28 of which showed more than 1.5-fold different intensity (P < 0.05) between the AS-C and AS-N samples. Proteins were identified in 17 out of the 28 spots. Fibrinogen and lumican were identified in 9 and 3 spots, respectively. Intensity of these 12 spots was lower in the AS-C samples than in the AS-N samples. In the 1D-Western blot analysis, 4 lumican bands (80 kDa, 75 kDa, 65 kDa, and 53 kDa) were detected, of which 2 bands with 80 kDa and 75 kDa showed lower intensity in the AS-C samples than in the AS-N samples. When de-glycosylated protein samples were used in the 1D-Western blot, only a single lumican band with ~40 kDa was detected, indicating that lumican was variously glycosylated and that highly glycosylated lumican molecules were decreased in AS-C.Collectively, insufficient glycosylation of lumican in the thickened and calcified areas of AS aortic valves may be involved in the pathophysiology of AS.
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Estenosis de la Válvula Aórtica/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Sulfato de Queratano/metabolismo , Proteómica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Electroforesis en Gel Bidimensional , Femenino , Glicosilación , Humanos , Lumican , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: To elucidate effects of salazosulfapyridine (SASP) and methotrexate (MTX), major anti-rheumatic drugs, on exosomes derived from SW982 of a human synovial sarcoma cell line. EXPERIMENTAL DESIGN: SW982 was treated with SASP and/or MTX under interleukin-1ß (IL-1ß)-treated or nontreated conditions. Exosomes were isolated from the culture media, and exosomal proteome was analyzed by 2D-DIGE. Protein spots whose intensity was significantly altered by the above treatments were identified by MS. RESULTS: Two hundred ninety-four protein spots were detected in the exosome preparations by 2D-DIGE. Compared to the nontreated cells, SASP-, MTX-, and (SASP + MTX)-treated cells displayed 8, 10, and 21 exosomal protein spots with more than ±2.0-fold intensity differences (p < 0.05), respectively. Similarly, the IL-1ß-treated cells displayed 58 exosomal protein spots with more than ±1.5-fold intensity differences (p < 0.05). In about half of the 58 spots, the IL-1ß-induced intensity changes were suppressed by simultaneous addition of SASP and/or MTX. Most of the identified proteins were immunity- or anti-oxidation-related proteins. CONCLUSIONS AND CLINICAL RELEVANCE: The SASP and/or MTX treatments altered the protein profiles of exosomes and suppressed the effects of IL-1ß on the exosomal proteome. Exosomes may play roles in the actions of these anti-rheumatic drugs.
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Antirreumáticos/farmacología , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Metotrexato/farmacología , Proteínas de Neoplasias/metabolismo , Sulfasalazina/farmacología , Electroforesis en Gel Bidimensional , Humanos , Interleucina-1beta/metabolismo , Proteínas de Neoplasias/análisis , Células Tumorales CultivadasRESUMEN
BACKGROUND: Tumor necrosis factor (TNF)-α is suggested to induce epithelial-mesenchymal transformation (EMT) of renal tubular epithelial cells that possibly exacerbates renal interstitial fibrosis in glomerulonephritis (GN). We here investigated whether layilin (LAYN), a c-type lectin-homologous protein, was involved in the EMT process. METHODS: Expression of LAYN was investigated in kidneys of mice administered with TNF-α and in a clear cell renal carcinoma cell line of KMRC-1 stimulated with TNF-α by quantitative polymerase chain reaction (qPCR) and/or western blotting. Expression of LAYN was assessed immunohistochemically in renal biopsy samples of patients with various types of GN. Changes of EMT markers and cell morphology by TNF-α and transforming growth factor (TGF)-ß in LAYN-knocked down KMRC-1 cells were investigated by qPCR and immunocytochemistry. RESULTS: Administration of TNF-α increased expression of LAYN in renal tubular epithelia in mice. TNF-α but not TGF-ß increased expression of LAYN in KMRC-1 cells. Renal biopsy samples from the patients with GN showed high expression of LAYN in tubular epithelial cells. TNF-α induced up-regulation of vimentin, down-regulation of E-cadherin, and fibroblast-like morphological change in KMRC-1 cells, indicating occurrence of EMT. These changes were not observed in the LAYN-knocked down cells. In contrast, similarly occurred TGF-ß-induced EMT was not affected by the LAYN knockdown. CONCLUSION: Our data indicate that LAYN is involved in the TNF-α-induced EMT of renal tubular epithelial cells. LAYN may play roles in the generation of renal interstitial fibrosis in GN via TNF-α-induced EMT.
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Proteínas Portadoras/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Túbulos Renales/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Glomerulonefritis/metabolismo , Humanos , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/genética , Masculino , Ratones , Ratones Endogámicos ICR , Factor de Necrosis Tumoral alfa/farmacología , Adulto JovenRESUMEN
OBJECTIVE: For diagnosis of dementia with Lewy bodies (DLB), we tried to find blood biomarkers for the disease. METHODS: Serum peptides were comprehensively detected by mass spectrometry. Peptides of interest were identified by tandem mass spectrometry. RESULTS: One hundred forty-six peptides were detected in a training set consisting of 30 DLB patients, 30 patients with Alzheimer's disease (AD), and 28 healthy control (HC) subjects. Multivariate analysis for discriminating the DLB group from the non-DLB (AD and HC) group using ion intensity of four peptides (2898, 4052, 4090, and 5002 m/z) showed sensitivity of 93.3% and specificity of 87.9% (DLB/nonDLB-4P model). In a testing set consisting of 20 DLB patients, 30 AD patients, and 14 HC subjects, this model showed sensitivity of 90.0% and specificity of 88.6%. DLB/nonDLB-4P model detected 86.7% and 90.0% of the AD patients as non-DLB in the training and testing sets, respectively, and discriminated all the 15 patients with amnestic mild cognitive impairment as non-DLB. Notably, a combination of two peptides (1737 and 5002 m/z) showed sensitivity of 95.0% and specificity of 93.3% for discriminating the DLB group from the AD group (DLB/nonDLB-2P model) in the testing set. The peptides used in these models included fragments from complement 4b, Wnt-2b, and lipopolysaccharide-binding protein, which were reported to be involved in the pathology of DLB or Parkinson's disease and hippocampal neurogenesis. CONCLUSIONS: Serum peptide profiles would provide useful DLB biomarker candidates, which may be implicated in the pathophysiology of the disease.
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Enfermedad de Alzheimer/sangre , Enfermedad por Cuerpos de Lewy/sangre , Péptidos/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Análisis Multivariante , Fragmentos de Péptidos/sangre , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. METHODS: Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. RESULTS: We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). CONCLUSION: Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.
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AMP Desaminasa/genética , Condrocitos/efectos de los fármacos , Regulación de la Expresión Génica , Ácidos Nucleicos/metabolismo , Osteoartritis de la Rodilla/tratamiento farmacológico , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , ARN/genética , AMP Desaminasa/biosíntesis , Anciano , Anciano de 80 o más Años , Western Blotting , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Femenino , Humanos , Janus Quinasa 3/antagonistas & inhibidores , Masculino , Ácidos Nucleicos/efectos de los fármacos , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA). In rheumatoid arthritis (RA), inflammatory cytokines like tumor necrosis factor α (TNF-α) have been known to play pathological roles. HA with low molecular weight is speculated to exacerbate inflammation in RA. In this context, differences of quantity and functions of HA receptors would affect the severity of inflammation in RA. Chondrocytes, which play critical roles in maintaining articular cartilage and are affected in RA, express at least kinds of HA receptors like CD44 and LAYN. However, roles and regulation of LAYN in articular chondrocytes have been poorly understood. To clarify regulation of LAYN in chondrocytes, we here investigated whether TNF-α affected expression levels of LAYN in human articular chondrocytes. Next, to clarify LAYN-specific roles in chondrocytes, we investigated whether binding of antibodies to the extracellular domain of LAYN affected secretion of inflammatory cytokines using a chondrosarcoma cell line. As a result, we found that TNF-α up-regulated expression levels of LAYN in the chondrocytes. Further, the LAYN signaling was found to enhance secretion of inflammatory factors, IL-8 and complement5 (C5)/C5a, from the cells. Our results indicate that LAYN would be involved in the enhancement of inflammation and degradation of cartilage in joint diseases such as RA and OA.
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Condrocitos/metabolismo , Mediadores de Inflamación/metabolismo , Lectinas Tipo C/metabolismo , Transducción de Señal , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: To promote understanding of immunoglobulin A nephropathy (IgAN) pathophysiology, we tried to elucidate glomerular protein profiles in IgAN, using microsieving that we established recently to isolate glomeruli from renal biopsy samples and proteomic approaches. METHODS: Glomeruli were isolated from renal biopsy samples of patients with IgAN (n = 5) and with minimal change nephrotic syndrome (MCNS; n = 5) using microsieving. Proteins extracted from the isolated glomeruli were separated by 2-dimensional differential gel electrophoresis (2D-DIGE). Proteins with different amounts between the two groups were identified by mass spectrometry. One of the identified proteins, α-actinin-4 (ACTN4), was further analyzed by Western blotting, RT-polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: By 2D-DIGE, 72 out of the detected 1,170 protein spots showed significantly different intensity between the two groups (p < 0.05). Thirty-four out of the 72 protein spots showed more than 1.5-fold or less than 1/1.5-fold intensity, out of which 16 protein spots were successfully identified. No microbial protein was identified. ACTN4 molecules with a low molecular weight of approximately 77 kDa were found to increase in the IgAN group. Lack of an N-terminal part of ACTN4 was demonstrated by Western blotting. No defect of mRNA for ACTN4 was evidenced by RT-PCR. Predominant existence of ACTN4 in capillary walls of glomeruli of IgAN patients was demonstrated by immunohistochemistry in glomerular sections of patients with IgAN. CONCLUSION: Use of microsieving enabled us to biochemically analyze glomerular proteins in renal biopsy samples from patients with glomerular diseases. With this method, we demonstrated skewed glomerular protein profiles in IgAN.
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Biopsia/métodos , Glomerulonefritis por IGA/inmunología , Glomérulos Renales/metabolismo , Proteómica/métodos , Actinina/química , Adolescente , Adulto , Electroforesis en Gel Bidimensional , Femenino , Humanos , Riñón/patología , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Nefrosis Lipoidea/inmunología , Estructura Terciaria de Proteína , Adulto JovenRESUMEN
OBJECTIVE: Sulfasalazine (SSZ) and tofacitinib are effective for treating rheumatoid arthritis, however, their effects on chondrocytes have not been fully understood. We here tried to elucidate their effects on chondrocyte proteins. METHODS: We treated chondrocytes from five osteoarthritis patients with IL-1ß, IL-1ß+ SSZ, IL-1ß+ tofacitinib, SSZ alone, and tofacitinib alone. Then, we compared protein profiles of the chondrocytes using two-dimensional differential gel electrophoresis. Further, we identified altered proteins by mass spectrometry. RESULTS: Out of 892 detected protein spots, the IL-1ß stimulation changed intensity of 43 spots more than 1.3-fold or less than 1/1.3-fold significantly. SSZ suppressed the IL-1ß-induced intensity alteration in 16 (37%) out of the 43 protein spots. Tofacitinib suppressed the IL-1ß-induced alteration in 4 (9.3%) out of the 43 spots. The production of AMP deaminase 2 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 were increased by IL-1ß and the increase was suppressed by SSZ and by tofacitinib. SSZ alone altered intensity of 273 (31%) out of the 852 spots significantly, whereas tofacitinib alone altered intensity of only 24 (2.7%) out of them. CONCLUSION: SSZ and, to lesser extent, tofacitinib suppress the effects of IL-1ß on the protein profiles of chondrocytes. Our data would promote understanding of effects of the drugs on chondrocytes.
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Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Sulfasalazina/farmacología , Anciano , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Femenino , Humanos , Interleucina-1beta/farmacología , Masculino , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVES: To investigate the pathophysiology of Behçet's disease (BD) and find biomarkers for the disease, we analysed protein profiles of peripheral blood mononuclear cells (PBMCs). METHODS: Proteins, extracted from PBMCs, were comprehensively analysed in 16 patients with BD, 16 patients with rheumatoid arthritis (RA), 12 patients with Crohn's disease (CD), and 16 healthy control subjects (HC) by 2-dimensional differential gel electrophoResis (2D-DIGE). Differently expressed proteins were identified by mass spectrometry. RESULTS: 563 protein spots were detected. We completely discriminated between the BD and HC groups, between the BD and RA groups, and between the BD and CD groups by multivariate analysis of intensity of 23, 35, and 1 spots, respectively. The spots contributing to the differences included proteins related to cytoskeleton, transcription/translation, T cell activation, bone turnover, regulating apoptosis, and microbial infection. Intensity of 3 spots (tyrosine-protein phosphatase non-receptor type 4, threonine synthase-like 2, and ß-actin) provided area under the receiver operating characteristic curves (AUROC) of 0.889 for discrimination between the BD group and the non-BD groups. Informatively, intensity of the above 1 spot completely discriminated the CD group from the other groups (AUROC 1.000). This spot, identified as ß-actin, had different pI from the above ß-actin-spot probably due to different post-translational modification. CONCLUSIONS: PBMC protein profiles, especially the profile of the 3 spots, would be candidate biomarkers for BD. The latter ß-actin subtype would be useful for discriminating inflammatory bowel diseases from BD and other diseases. The identified proteins may play important roles in the pathophysiology of BD.
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Síndrome de Behçet/diagnóstico , Síndrome de Behçet/metabolismo , Leucocitos Mononucleares/metabolismo , Proteómica/métodos , Electroforesis Bidimensional Diferencial en Gel/métodos , Adolescente , Adulto , Anciano , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Síndrome de Behçet/inmunología , Biomarcadores/metabolismo , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana EdadRESUMEN
OBJECTIVE: To find a blood biomarker and disease-related peptides in Alzheimer's disease (AD), we comprehensively detected serum peptides. METHODS: Ion intensity of serum peptides from 62 AD patients and 82 control subjects was measured by mass spectrometry. RESULTS: A total of 157 peptides were detected from 30 AD patients and 30 healthy control (HC) subjects. Sixty out of the 157 peptide profiles discriminated between the AD and HC groups. Sixteen out of the 60 peptides were identified, 10 out of which were fragments of a fibrinogen α chain (FIBA). Among the 10 peptides, four and six peptides were derived from fibrinopeptide A (FPA, Aα1-16) and the C-terminal region of the αC-domain (αCDC, Aα557-610), respectively. The profile of 10 FIBA-derived peptides combined with age discriminated between the two groups with an area under the receiver operating characteristic curve (AUROC) of 0.940. Validation of this model using a testing set of 32 AD patients and 19 HC subjects showed an AUROC of 0.717, sensitivity of 65.6%, and specificity of 73.7% by a cutoff value of 0.56420. Another value, 0.04029, showed sensitivity of 96.9%, suggesting that subjects with values less than 0.04029 rarely possess AD. FPA and αCDC showed increased ion intensity in the AD group compared with the HC group (p < 0.05). CONCLUSIONS: The profile of 10 FIBA-derived peptides combined with age would be a candidate biomarker for AD, which facilitates screening of the disease. The significant release of FPA and αCDC may be involved in the aberrant coagulation that leads to vascular damage in AD.
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Enfermedad de Alzheimer/sangre , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinógeno/análisis , Fragmentos de Péptidos/sangre , Factores de Edad , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Curva ROC , Sensibilidad y EspecificidadRESUMEN
Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p<0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. BIOLOGICAL SIGNIFICANCE: In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA.