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Identifying molecular mediators of neural circuit development and/or function that contribute to circuit dysfunction when aberrantly reengaged in neurological disorders is of high importance. The role of the TWEAK/Fn14 pathway, which was recently reported to be a microglial/neuronal axis mediating synaptic refinement in experience-dependent visual development, has not been explored in synaptic function within the mature central nervous system. By combining electrophysiological and phosphoproteomic approaches, we show that TWEAK acutely dampens basal synaptic transmission and plasticity through neuronal Fn14 and impacts the phosphorylation state of pre- and postsynaptic proteins in adult mouse hippocampal slices. Importantly, this is relevant in two models featuring synaptic deficits. Blocking TWEAK/Fn14 signaling augments synaptic function in hippocampal slices from amyloid-beta-overexpressing mice. After stroke, genetic or pharmacological inhibition of TWEAK/Fn14 signaling augments basal synaptic transmission and normalizes plasticity. Our data support a glial/neuronal axis that critically modifies synaptic physiology and pathophysiology in different contexts in the mature brain and may be a therapeutic target for improving neurophysiological outcomes.
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Degeneración Nerviosa/metabolismo , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Sinapsis/metabolismo , Receptor de TWEAK/metabolismo , Animales , Citocina TWEAK/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipocampo/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/fisiopatología , Plasticidad Neuronal/fisiología , Terminales Presinápticos/metabolismo , Accidente Cerebrovascular/fisiopatología , Transmisión Sináptica/fisiologíaRESUMEN
SUMMARY: We developed Quickomics, a feature-rich R Shiny-powered tool to enable biologists to fully explore complex omics statistical analysis results and perform advanced analysis in an easy-to-use interactive interface. It covers a broad range of secondary and tertiary analytical tasks after primary analysis of omics data is completed. Each functional module is equipped with customizable options and generates both interactive and publication-ready plots to uncover biological insights from data. The modular design makes the tool extensible with ease. AVAILABILITY AND IMPLEMENTATION: Researchers can experience the functionalities with their own data or demo RNA-Seq and proteomics datasets by using the app hosted at http://quickomics.bxgenomics.com and following the tutorial, https://bit.ly/3rXIyhL. The source code under GPLv3 license is provided at https://github.com/interactivereport/Quickomics for local installation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Novel therapies and biomarkers are needed for the treatment of acute ischemic stroke (AIS). This study aimed to provide comprehensive insights into the dynamic proteome changes and underlying molecular mechanisms post-ischemic stroke. TMT-coupled proteomic analysis was conducted on mouse brain cortex tissue from five time points up to 4 weeks poststroke in the distal hypoxic-middle cerebral artery occlusion (DH-MCAO) model. We found that nearly half of the detected proteome was altered following stroke, but only â¼8.6% of the changes were at relatively large scales. Clustering on the changed proteome defined four distinct expression patterns characterized by temporal and quantitative changes in innate and adaptive immune response pathways and cytoskeletal and neuronal remodeling. Further analysis on a subset of 309 "top hits", which temporally responded to stroke with relatively large and sustained changes, revealed that they were mostly secreted proteins, highly correlated to different cortical cytokines, and thereby potential pharmacodynamic biomarker candidates for inflammation-targeting therapies. Closer examination of the top enriched neurophysiologic pathways identified 57 proteins potentially associated with poststroke recovery. Altogether, our study generated a rich dataset with candidate proteins worthy of further validation as biomarkers and/or therapeutic targets for stroke. The proteomics data are available in the PRIDE Archive with identifier PXD025077.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Ratones , Proteoma/genética , ProteómicaRESUMEN
Tau-tubulin kinase 1 (TTBK1) is a CNS-specific, kinase that has been implicated in the pathological phosphorylation of tau in Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD). TTBK1 is a challenging therapeutic target because it shares a highly conserved catalytic domain with its homolog, TTBK2, a ubiquitously expressed kinase genetically linked to the disease spinocerebellar ataxia type 11. The present study attempts to elucidate the functional distinctions between the TTBK isoforms and increase our understanding of them as distinct targets for the treatment of neurodegenerative disease. We demonstrate that in cortical neurons, TTBK1, not TTBK2, is the isoform responsible for tau phosphorylation at epitopes enriched in tauopathies such as Serine 422. In addition, although our elucidation of the crystal structure of the TTBK2 kinase domain indicates almost identical structural similarity with TTBK1, biochemical and cellular assays demonstrate that the enzymatic activity of these two proteins is regulated by a combination of unique extra-catalytic sequences and autophosphorylation events. Finally, we have identified an unbiased list of neuronal interactors and phosphorylation substrates for TTBK1 and TTBK2 that highlight the unique cellular pathways and functional networks that each isoform is involved in. This data address an important gap in knowledge regarding the implications of targeting TTBK kinases and may prove valuable in the development of potential therapies for disease.
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Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Corteza Cerebral/patología , Epítopos/metabolismo , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/química , Proteómica , Serina/metabolismo , Homología Estructural de Proteína , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. METHODS: A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. RESULTS: Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. CONCLUSIONS: Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.
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Neuronas Dopaminérgicas/metabolismo , Endocitosis/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación , Animales , Perfilación de la Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteómica , Ratas , Ratas Transgénicas , Vesículas Sinápticas/genéticaRESUMEN
Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/ß-catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis.
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Factor de Crecimiento del Tejido Conjuntivo/fisiología , Enfermedades Renales/etiología , Riñón/patología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Animales , Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Fibroblastos , Fibrosis/etiología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , PericitosRESUMEN
BACKGROUND: The excessive accumulation of extracellular matrix (ECM) in the renal tubulointerstitium is a key component of chronic renal damage in lupus nephritis (LN) and a critical determinant of the disease progression to renal failure. Detection of fibrosis requires renal biopsy and is therefore limited by high risks associated with an invasive procedure. This study explores whether a unique LN urinary peptidome can be identified and whether LN-specific alteration reflects the underlying fibrogenic process of altered ECM turnover. METHOD: Urinary peptides were analyzed for 36 LN and 35 nonrenal systemic lupus erythematosus (SLE) subjects and 58 healthy volunteers (HVs). RESULTS: In total, 70 collagen and 230 noncollagen peptides were significantly changed between LN and nonrenal SLE and between LN and HV and defined as 'LN peptides'; 14 proteases associated with observed LN collagen peptides were identified and activities in 9 proteases were significantly different between LN and nonrenal SLE; 28 collagen peptides were correlated with at least one parameter of clinical renal dysfunction or histolopathology. CONCLUSION: Urinary peptidomic alterations likely reflect pathogenic pathways involving ECM turnover in LN kidneys and potentially could be developed as biomarkers to monitor renal disease progression.
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Biomarcadores/orina , Colágeno/orina , Enfermedades Renales/diagnóstico , Lupus Eritematoso Sistémico/complicaciones , Nefritis Lúpica/complicaciones , Fragmentos de Péptidos/orina , Adulto , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Humanos , Enfermedades Renales/etiología , Enfermedades Renales/orina , MasculinoRESUMEN
Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.
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Permeabilidad Capilar/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Anhidrasas Carbónicas/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/metabolismo , Sistema Calicreína-Quinina/fisiología , Cuerpo Vítreo/enzimología , Acetazolamida/farmacología , Animales , Western Blotting , Antagonistas de los Receptores de Bradiquinina , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/toxicidad , Complemento C1/antagonistas & inhibidores , Factor XIIa/metabolismo , Humanos , Espectrometría de Masas , Papiledema/inducido químicamente , Proteómica , Ratas , Ratas Sprague-Dawley , Estadísticas no ParamétricasRESUMEN
Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postnatal day 1 (p1) and 45 (p45), as a model system, we compared the protein expression ratios (p45/p1) observed using duplex TMT tags in triplicate measurements versus sixplex tag in a single LC-MS/MS analysis. A correlation of 0.79 in relative protein abundance was observed in the proteins quantified by these two sets of reagents. However, more proteins passed the criteria for significant fold change (-1.0 ≤ log(2) ratio (p45/p1) ≥ +1.0 and p < 0.05) in the sixplex analysis. Nevertheless, in both methods most proteins showing significant fold change were identified by multiple spectra, increasing their quantification precision. Additionally, the fold change in p45 rats against p1, observed in TMT experiments, was corroborated by a metabolic labeling strategy where relative quantification of differentially expressed proteins was obtained using (15)N-labeled p45 rats as an internal standard.
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Química Encefálica , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/análisis , Fragmentos de Péptidos/análisis , Coloración y Etiquetado/métodos , Animales , Animales Recién Nacidos , Cromatografía Liquida , Marcaje Isotópico , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/química , Proteómica , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Células Endoteliales/metabolismo , Insulina/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Animales , Bovinos , Células Cultivadas , Células Endoteliales/citología , Activación Enzimática/fisiología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Enfermedades Metabólicas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Colorectal cancer (CRC) is the third most common cancer worldwide and has poor prognosis. To identify the oncofetal proteins involved in CRC carcinogenesis, differentially expressed proteins among fetal colorectal tissues, CRC, and the paired tumor-adjacent normal colorectal tissues were investigated by a two-dimensional gel electrophoresis and MALDI-TOF/TOF-based proteomics approach. 42 protein spots were differentially expressed among these tissues, and 22 proteins were identified by MS analysis. Desmin and zinc finger protein 829 were found to be elevated in CRC tissue and fetal colorectal tissue compared with normal colorectal tissue. The elevated expression of desmin in CRC tissue and different developmental stages of fetus colon was confirmed by RT-PCR and Western blot analysis. Immunohistochemical analysis showed that the elevated expression of desmin was correlated with the severity and differentiation of CRC and decreased survival rate of CRC patients. Finally by developing a highly sensitive immunoassay, desmin could be detected in human serum and was significantly elevated in CRC patients compared with healthy volunteers. We propose that desmin be considered a potential oncofetal serum tumor marker for CRC that may have significance in the detection of patients with CRC.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/diagnóstico , Desmina/análisis , Enfermedades Fetales/diagnóstico , Proteómica/métodos , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Western Blotting , Colon/embriología , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Desmina/genética , Desmina/metabolismo , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedades Fetales/genética , Enfermedades Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.
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Angiotensina II/farmacología , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Proteoma/análisis , Coloración y Etiquetado , Animales , Western Blotting , Cromatografía Liquida , Biología Computacional , Medios de Cultivo Condicionados , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Diabetic retinopathy is the most common microvascular complication caused by diabetes mellitus and is a leading cause of vision loss among working-age adults in developed countries. Understanding the effects of diabetes on the retinal proteome may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of retina from C57BL/6 mice with 2 months of streptozotocin-induced diabetes and age-matched nondiabetic control mice. To explore the role of the angiotensin AT1 receptor in the retinal proteome in diabetes, a subgroup of mice were treated with the AT1 antagonist candesartan. We identified 1792 proteins from retinal lysates, of which 65 proteins were differentially changed more than 2-fold in diabetic mice compared with nondiabetic mice. A majority (72%) of these protein changes were normalized by candesartan treatment. Most of the significantly changed proteins were associated with metabolism, oxidative phosphorylation, and apoptotic pathways. An analysis of the proteomics data revealed metabolic and apoptotic abnormalities in the retina from diabetic mice that were ameliorated with candesartan treatment. These results provide insight into the effects of diabetes on the retina and the role of the AT1 receptor in modulating this response.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Retinopatía Diabética/metabolismo , Proteínas del Ojo/análisis , Proteoma/efectos de los fármacos , Retina/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Apoptosis , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Compuestos de Bifenilo , Diabetes Mellitus/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/metabolismo , Metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación Oxidativa , Proteómica , Estreptozocina , Tetrazoles/farmacología , Tetrazoles/uso terapéuticoRESUMEN
Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-ß, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct "fibrotic" matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression.
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Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.
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Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Regulación Neoplásica de la Expresión Génica , FN-kappa B/metabolismo , Osteosarcoma/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Ciclinas/metabolismo , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/farmacología , Receptor beta de Linfotoxina/antagonistas & inhibidores , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Propionatos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma , Transducción de Señal , Receptor de TWEAK/antagonistas & inhibidores , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismo , Células Tumorales Cultivadas , Quinasa de Factor Nuclear kappa BRESUMEN
PURPOSE: Originally identified as a lipopolysaccharide binding protein with Gram-negative bactericidal activity in the leukocytes, bactericidal/permeability-increasing protein (BPI) has been shown to induce various effects in retinal cells in vivo and in vitro. METHODS: The authors recently reported that BPI can induce ERK1/2 and Akt activity and that it increases DNA synthesis in the bovine retinal pigment epithelial (RPE) and pericyte cells. The authors have extended the characterization of BPI interaction with membrane proteins from bovine RPE. Crude membrane pools from RPE were isolated, solubilized, and bound to rBPI(21) affinity column. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue, which showed an intense band at 36 kDa consistently displaced by rBPI(21). RESULTS: Tandem mass spectrometry of the 36-kDa band suggested that cell surface protein glypican 4 (GPC4) serves as a putative BPI-binding protein. Heparitinase, phosphatidylinositol-specific phospholipase C, and anti-GPC4 antibody suppressed BPI-induced ERK and Akt phosphorylation in bovine RPE. Moreover, heparitinase also inhibited BPI actions on VEGF and PDGF-B mRNA expression induced by H(2)O(2). CONCLUSIONS: These new findings suggest that GPC4 is a specific binding protein for BPI on RPE to mediate the activation of ERK1/2, Akt, and the mRNA expressions of PDGF-B and VEGF.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Glipicanos/metabolismo , Proteínas de la Membrana/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Transducción de Señal/fisiología , Animales , Péptidos Catiónicos Antimicrobianos/química , Proteínas Sanguíneas/química , Bovinos , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Glipicanos/química , Immunoblotting , Proteínas de la Membrana/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Epitelio Pigmentado Ocular/efectos de los fármacos , Polisacárido Liasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-sis/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Fosfolipasas de Tipo C/farmacología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Bactericidal/permeability-increasing protein (BPI) was originally identified as a lipopolysaccharide (LPS) binding protein with gram-negative bactericidal activity in the leukocytes. In this study, we characterized the previously unknown effects of BPI in the eye and the molecular mechanisms involved in its action. BPI mRNA was detected in bovine retina; retinal pigment epithelium; and primary cultures of bovine retinal pigment epithelial cells (RPE), pericytes (RPC), and endothelial cells (REC); while BPI protein was measured in human vitreous and plasma. BPI, but not control protein thaumatin, activated extracellular regulated kinase (ERK) and AKT, and increased DNA synthesis in RPE and RPC but not in REC. A human recombinant 21 kDa modified amino-terminal fragment of BPI (rBPI21) reduced H2O2-induced apoptosis in RPE and inhibited vascular endothelial growth factor (VEGF)-stimulated ERK phosphorylation in REC when preincubated with VEGF. Intraperitoneal (i.p.)-injected rBPI21 reduced ischemia-induced retinal neovascularization and diabetes-induced retinal permeability. Since BPI has unusual dual properties of promoting RPC and RPE growth while suppressing VEGF-induced REC growth and vascular permeability, the mechanistic understanding of BPI's action may provide novel therapeutic opportunities for diabetic retinopathy and age-related macular degeneration.
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Inhibidores de la Angiogénesis/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Sanguíneas/farmacología , Proteínas de la Membrana/farmacología , Retina/citología , Vasos Retinianos/efectos de los fármacos , Transducción de Señal , Animales , Péptidos Catiónicos Antimicrobianos/análisis , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/análisis , Permeabilidad Capilar/efectos de los fármacos , Bovinos , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Proteínas de la Membrana/análisis , Neovascularización Patológica/tratamiento farmacológico , Pericitos/citología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/metabolismo , Plasma/química , Proteínas Recombinantes/farmacología , Retina/efectos de los fármacos , Retina/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/farmacología , Cuerpo Vítreo/químicaRESUMEN
PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.
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Angiotensina II/fisiología , Fosforilación , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiología , Angiotensina II/metabolismo , Animales , Línea Celular , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/químicaRESUMEN
BACKGROUND: Parkinson disease (PD) is a neurodegenerative disease characterized by the accumulation of alpha-synuclein (SNCA) and other proteins in aggregates termed "Lewy Bodies" within neurons. PD has both genetic and environmental risk factors, and while processes leading to aberrant protein aggregation are unknown, past work points to abnormal levels of SNCA and other proteins. Although several genome-wide studies have been performed for PD, these have focused on DNA sequence variants by genome-wide association studies (GWAS) and on RNA levels (microarray transcriptomics), while genome-wide proteomics analysis has been lacking. METHODS: This study employed two state-of-the-art technologies, three-stage Mass Spectrometry Tandem Mass Tag Proteomics (12 PD, 12 controls) and RNA-sequencing transcriptomics (29 PD, 44 controls), evaluated in the context of PD GWAS implicated loci and microarray transcriptomics (19 PD, 24 controls). The technologies applied for this study were performed in a set of overlapping prefrontal cortex (Brodmann area 9) samples obtained from PD patients and sex and age similar neurologically healthy controls. RESULTS: After appropriate filters, proteomics robustly identified 3558 unique proteins, with 283 of these (7.9 %) significantly different between PD and controls (q-value < 0.05). RNA-sequencing identified 17,580 protein-coding genes, with 1095 of these (6.2 %) significantly different (FDR p-value < 0.05); only 166 of the FDR significant protein-coding genes (0.94 %) were present among the 3558 proteins characterized. Of these 166, eight genes (4.8 %) were significant in both studies, with the same direction of effect. Functional enrichment analysis of the proteomics results strongly supports mitochondrial-related pathways, while comparable analysis of the RNA-sequencing results implicates protein folding pathways and metallothioneins. Ten of the implicated genes or proteins co-localized to GWAS loci. Evidence implicating SNCA was stronger in proteomics than in RNA-sequencing analyses. CONCLUSIONS: We report the largest analysis of proteomics in PD to date, and the first to combine this technology with RNA-sequencing to investigate GWAS implicated loci. Notably, differentially expressed protein-coding genes were more likely to not be characterized in the proteomics analysis, which lessens the ability to compare across platforms. Combining multiple genome-wide platforms offers novel insights into the pathological processes responsible for this disease by identifying pathways implicated across methodologies.
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Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , Pliegue de Proteína , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Ontología de Genes , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARNRESUMEN
Effects of scorpion venom active polypeptide (SVAP) from scorpion venom of Buthus Martensii Karsch of Chinese on platelet aggregation in ex vivo and vitro in rabbits, thrombosis in carotid artery of rats and plasma 6-keto-PG F1alpha and TXB2 in rats were studied by the turbidimetry, the duplicated thrombosis model by electrostimulation and RIA, respectively. The results showed that SVAP 0.125, 0.25, 0.5 mg/ml inhibited significantly the rabbit platelet aggregation triggered by 0.3 U/ml thrombin, 10 microM ADP in vitro (P<0.05 or 0.01) and SVAP at the dose of 0.32, 0.64 mg/kg iv prolonged distinctively the occlusion time of thrombosis that were induced by electrical stimulation. Increased% of 0.16, 0.32 and 0.64 mg/kg were 30.16, 71.74, 98.27%, respectively, which showed a good dose-effect relationship. SVAP 0.22 mg/ml (in vitro) or 0.2, 0.4 mg/kg (in ex vivo) could obviously increase the plasma concentration of 6-keto-PG F1alpha, but slightly effect rats plasma concentration of TXB2 in vitro and in ex vivo and significantly increase of value of PG I2/TXA2, which suggested that the mechanism of the antithrombotic action of SVAP is related to the resistance against platelet aggregation, increase of the concentration of PG I2 in plasma.