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1.
J Exp Clin Cancer Res ; 42(1): 201, 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37559126

RESUMO

BACKGROUND: The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches. METHODS AND RESULTS: Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5ß1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models. CONCLUSIONS: This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.


Assuntos
Carcinoma Ductal Pancreático , Fibronectinas , Neoplasias Pancreáticas , Pancreatite , Animais , Humanos , Camundongos , Doença Aguda , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Fibronectinas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Proteômica , Tripsina/metabolismo , Microambiente Tumoral , Neoplasias Pancreáticas
2.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34047769

RESUMO

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.


Assuntos
Nadadeiras de Animais/metabolismo , Movimento Celular , Inflamação/metabolismo , Interleucinas/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Cicatrização , Infecção dos Ferimentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/lesões , Nadadeiras de Animais/microbiologia , Nadadeiras de Animais/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/microbiologia , Interleucinas/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Microscopia de Fluorescência , Neutrófilos/microbiologia , Comunicação Parácrina , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Fatores de Tempo , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/microbiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
3.
Angiogenesis ; 22(1): 133-144, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30168023

RESUMO

Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.


Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Ressonância de Plasmônio de Superfície , Trombospondinas/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Sequências Repetitivas de Aminoácidos , Trombospondinas/metabolismo
4.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30396995

RESUMO

Control of synapse number and function in the developing central nervous system is critical to the formation of neural circuits. Astrocytes play a key role in this process by releasing factors that promote the formation of excitatory synapses. Astrocyte-secreted thrombospondins (TSPs) induce the formation of structural synapses, which however remain post-synaptically silent, suggesting that completion of early synaptogenesis may require a two-step mechanism. Here, we show that the humoral innate immune molecule Pentraxin 3 (PTX3) is expressed in the developing rodent brain. PTX3 plays a key role in promoting functionally-active CNS synapses, by increasing the surface levels and synaptic clustering of AMPA glutamate receptors. This process involves tumor necrosis factor-induced protein 6 (TSG6), remodeling of the perineuronal network, and a ß1-integrin/ERK pathway. Furthermore, PTX3 activity is regulated by TSP1, which directly interacts with the N-terminal region of PTX3. These data unveil a fundamental role of PTX3 in promoting the first wave of synaptogenesis, and show that interplay of TSP1 and PTX3 sets the proper balance between synaptic growth and synapse function in the developing brain.


Assuntos
Proteína C-Reativa/fisiologia , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Astrócitos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteína C-Reativa/genética , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Matriz Extracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Transporte Proteico/genética , Trombospondina 1/metabolismo
5.
PLoS One ; 13(7): e0201320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30048528

RESUMO

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.


Assuntos
Moléculas de Adesão Celular/imunologia , Eosinófilos/citologia , Proteínas da Matriz Extracelular/imunologia , Fator de Crescimento Transformador beta/imunologia , Adesão Celular , Ensaios de Migração de Leucócitos , Movimento Celular , Eosinófilos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-3/imunologia , Interleucina-5/imunologia
6.
J Biol Chem ; 293(34): 13166-13175, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-29954947

RESUMO

Human myeloid-derived growth factor (MYDGF; also known as C19orf10) is named based on its identification as a secreted monocyte/macrophage-derived mediator of cardiac repair following myocardial infarction in mice. Homologs of MYDGF, however, are present in organisms throughout and outside of the animal kingdom, some of which lack hematopoietic and circulatory systems. Moreover, the UPF0556 protein domain, which defines these homologs, lacks a known structure. As a result, the functions and properties of MYDGF are unclear. Our current work was initiated to test whether MYDGF is present in secretory vesicles of eosinophils as it was recently reported to be abundant in these cells. However, we could not demonstrate secretion and unexpectedly discovered that MYDGF colocalizes with P4HB in the nuclear envelope, which comprises the bulk of endoplasmic reticulum (ER) in eosinophils, and with P4HB and RCAS1 in Golgi. We noted a ubiquitous C-terminal sequence, BXEL (B, basic; X, variable residue; E, Glu; L, Leu), that has the potential to retain human MYDGF and its homologs in the ER. To test the functionality of this sequence, we expressed full-length human MYDGF or MYDGF lacking the C-terminal Glu-Leu residues in monolayers of human embryonic kidney 293 (HEK293) cells. Full-length MYDGF accumulated in cells, whereas truncated MYDGF appeared in the medium. These observations reveal that MYDGF resides in the ER and Golgi and provide a new framework for investigating and understanding this intriguing protein.


Assuntos
Antígenos de Neoplasias/metabolismo , Retículo Endoplasmático/metabolismo , Eosinófilos/metabolismo , Complexo de Golgi/metabolismo , Interleucinas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Transporte Biológico , Humanos , Interleucinas/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Isomerases de Dissulfetos de Proteínas/genética , Homologia de Sequência
7.
Mol Cell Proteomics ; 17(7): 1410-1425, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29669734

RESUMO

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.


Assuntos
Proteínas ADAMTS/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Morfogênese , Proteólise , Proteômica/métodos , Esferoides Celulares/metabolismo , Células 3T3 , Proteínas ADAMTS/química , Sequência de Aminoácidos , Animais , Colágeno/metabolismo , Cães , Fibroblastos/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Regulação para Cima
8.
Am J Respir Crit Care Med ; 196(11): 1385-1395, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28862877

RESUMO

RATIONALE: Mepolizumab, an IL-5-blocking antibody, reduces exacerbations in patients with severe eosinophilic asthma. Mepolizumab arrests eosinophil maturation; however, the functional phenotype of eosinophils that persist in the blood and airway after administration of IL-5 neutralizing antibodies has not been reported. OBJECTIVES: To determine the effect of anti-IL-5 antibody on the numbers and phenotypes of allergen-induced circulating and airway eosinophils. METHODS: Airway inflammation was elicited in participants with mild allergic asthma by segmental allergen challenge before and 1 month after a single intravenous 750-mg dose of mepolizumab. Eosinophils were examined in blood, bronchoalveolar lavage, and endobronchial biopsies 48 hours after challenge. MEASUREMENTS AND MAIN RESULTS: Segmental challenge without mepolizumab induced a rise in circulating eosinophils, bronchoalveolar lavage eosinophilia, and eosinophil peroxidase deposition in bronchial mucosa. IL-5 neutralization before allergen challenge abolished the allergen-induced rise in circulating eosinophils and expression of IL-3 receptors, whereas airway eosinophilia and eosinophil peroxidase deposition were blunted but not eliminated. Before mepolizumab treatment, bronchoalveolar lavage eosinophils had more surface IL-3 and granulocyte-monocyte colony-stimulating factor receptors, CD69, CD44, and CD23 and decreased IL-5 and eotaxin receptors than blood eosinophils. This activation phenotype indicated by bronchoalveolar lavage eosinophil surface markers, as well as the release of eosinophil peroxidase by eosinophils in the bronchial mucosa, was maintained after mepolizumab. CONCLUSIONS: Mepolizumab reduced airway eosinophil numbers but had a limited effect on airway eosinophil activation markers, suggesting that these cells retain functionality. This observation may explain why IL-5 neutralization reduces but does not completely eradicate asthma exacerbations. Clinical trial registered with www.clinicaltrials.gov (NCT00802438).


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Asma/metabolismo , Brônquios/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/metabolismo , Adulto , Asma/patologia , Biópsia , Brônquios/diagnóstico por imagem , Líquido da Lavagem Broncoalveolar , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase
9.
Acta Haematol ; 136(2): 85-97, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27188909

RESUMO

BACKGROUND/AIMS: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells. METHODS: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells. RESULTS: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts. CONCLUSIONS: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts.


Assuntos
Diferenciação Celular , Células Cultivadas , Proliferação de Células , Fibroblastos/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia
10.
J Proteome Res ; 15(5): 1524-33, 2016 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-27005946

RESUMO

A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of platelets that routinely contaminate purified eosinophils and report the variability in the eosinophil proteome among five individuals and proteomic changes accompanying acute activation of eosinophils by interleukin-5. Our deep coverage and quantitative analyses fill an important gap in the existing maps of the human proteome and will enable the strategic use of proteomics to study eosinophils in human diseases.


Assuntos
Eosinófilos/química , Proteoma/análise , Cromatografia Líquida/métodos , Humanos , Interleucina-5/farmacologia , Espectrometria de Massas/métodos , Fosforilação , Proteômica/métodos
11.
Crit Rev Biochem Mol Biol ; 50(5): 427-39, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26288337

RESUMO

Periostin (PN) and TGF-ß-induced protein (ßig-h3) are paralogs that contain a single emilin and four fasciclin-1 modules and are secreted from cells. PN receives attention because of its up-regulation in cancer and degenerative and allergic diseases. ßig-h3 is highly enriched in cornea and best known for harboring mutations in humans associated with corneal dystrophies. Both proteins are expressed widely, and many functions, some over-lapping, have been attributed to PN and ßig-h3 based on biochemical, cell culture, and whole animal experiments. We attempt to organize this knowledge so as to facilitate research on these interesting and incompletely understood proteins. We focus particularly on whether PN and ßig-h3 are modified by vitamin K-dependent γ-glutamyl carboxylation, a question of considerable importance given the profound effects of γ-carboxylation on structure and function of other proteins. We consider the roles of PN and ßig-h3 in formation of extracellular matrix and as ligands for integrin receptors. We attempt to reconcile the contradictory results that have arisen concerning the role of PN, which has emerged as a marker of TH2 immunity, in murine models of allergic asthma. Finally, when possible we compare and contrast the structures and functions of the two proteins.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Imunidade Celular , Integrinas/agonistas , Modelos Moleculares , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Sequência Conservada , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Humanos , Integrinas/metabolismo , Ligantes , Filogenia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética
12.
Circ Res ; 117(2): 129-41, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-25940549

RESUMO

RATIONALE: Histological examination of abdominal aortic aneurysm (AAA) tissues demonstrates extracellular matrix destruction and infiltration of inflammatory cells. Previous work with mouse models of AAA has shown that anti-inflammatory strategies can effectively attenuate aneurysm formation. Thrombospondin-1 is a matricellular protein involved in the maintenance of vascular structure and homeostasis through the regulation of biological functions, such as cell proliferation, apoptosis, and adhesion. Expression levels of thrombospondin-1 correlate with vascular disease conditions. OBJECTIVE: To use thrombospondin-1-deficient (Thbs1(-/-)) mice to test the hypothesis that thrombospondin-1 contributes to pathogenesis of AAAs. METHODS AND RESULTS: Mouse experimental AAA was induced through perivascular treatment with calcium phosphate, intraluminal perfusion with porcine elastase, or systemic administration of angiotensin II. Induction of AAA increased thrombospondin-1 expression in aortas of C57BL/6 or apoE-/- mice. Compared with Thbs1(+/+) mice, Thbs1(-/-) mice developed significantly smaller aortic expansion when subjected to AAA inductions, which was associated with diminished infiltration of macrophages. Thbs1(-/-) monocytic cells had reduced adhesion and migratory capacity in vitro compared with wild-type counterparts. Adoptive transfer of Thbs1(+/+) monocytic cells or bone marrow reconstitution rescued aneurysm development in Thbs1(-/-) mice. CONCLUSIONS: Thrombospondin-1 expression plays a significant role in regulation of migration and adhesion of mononuclear cells, contributing to vascular inflammation during AAA development.


Assuntos
Aneurisma da Aorta Abdominal/fisiopatologia , Macrófagos/fisiologia , Trombospondina 1/fisiologia , Transferência Adotiva , Angiotensina II/toxicidade , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Transplante de Medula Óssea , Fosfatos de Cálcio/toxicidade , Linhagem Celular , Movimento Celular , Modelos Animais de Doenças , Inflamação , Macrófagos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologia , Monócitos/transplante , Elastase Pancreática/toxicidade , Quimera por Radiação , Proteínas Recombinantes/uso terapêutico , Trombospondina 1/biossíntese , Trombospondina 1/deficiência , Trombospondina 1/uso terapêutico , Regulação para Cima
13.
PLoS One ; 10(5): e0127243, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25984943

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3ß, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-ß, and ΔS-ß) rather than the three reported in publically available databases from which ΔS-ß is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-ß. ΔS-α and ΔS-ß together comprised 15.6 ± 4.0 % (mean ± SD, n = 21) of the total. The percentage of STAT3ß variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina's "BodyMap" RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-ß, and ΔS-ß variants of STAT3.


Assuntos
Eosinófilos/metabolismo , Linfoma Difuso de Grandes Células B/genética , Fator de Transcrição STAT3/genética , Processamento Alternativo , Sequência de Aminoácidos , Linfócitos B/patologia , Sequência de Bases , Linhagem Celular Tumoral , Centro Germinativo/patologia , Humanos , Dados de Sequência Molecular , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fator de Transcrição STAT3/química , Transcrição Gênica
14.
PLoS One ; 10(4): e0124941, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25919138

RESUMO

Intrinsically disordered sequences within bacterial adhesins bind to E-strands in the ß-sheets of multiple FNI modules of fibronectin (FN) by anti-parallel ß-strand addition, also called tandem ß-zipper formation. The FUD segment of SfbI of Streptococcus pyogenes and Bbk32 segment of BBK32 of Borrelia burgdorferi, despite being imbedded in different adhesins from different bacteria, target the same 2-5,8-9 FNI modules, 2-5,8-9 FNI, in the N-terminal 70-kDa region (FN70K) of FN. To facilitate further comparisons, FUD, Bbk32, two other polypeptides based on SfbI that target 1-5 FNI (HADD) and 2-5 FNI (FRD), and mutant Bbk32 (ΔBbk32) were produced with fluorochromes placed just outside of the binding sequences. Unlabeled FUD competed ~ 1000-fold better for binding of labeled Bbk32 to FN than unlabeled Bbk32 competed for binding of labeled FUD to FN. Binding kinetics were determined by fluorescence polarization in a stopped-flow apparatus. On-rates for FUD, Bbk32, HADD, and FRD were similar, and all bound more rapidly to FN70K fragment than to full length FN. In stopped-flow displacement and size exclusion chromatographic assays, however, k off for FUD or HADD to FN70K or FN was considerably lower compared to k off of FRD or Bbk32. FUD and Bbk32 differ in the spacing between sequences that interact with 3FNI and 4FNI or with 5FNI and 8FNI. ΔBbk32, in which 2 residues were removed from Bbk32 to make the spacing more like FUD, had a k off intermediate between that of Bbk32 and FUD. These results indicate a "folding-after-binding" process after initial association of certain polypeptide sequences to FN that results in formation of a stable complex and is a function of number of FNI modules engaged by the polypeptide, spacing of engagement sites, and perhaps flexibility within the polypeptide-FN complex. We suggest that contributions of SfbI and BBK32 adhesins to bacterial pathogenicity may be determined in part by stability of adhesin-FN complexes.


Assuntos
Fibronectinas/química , Fibronectinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Fluorometria , Cinética , Ligantes , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Solubilidade
15.
J Biol Chem ; 290(8): 4866-4876, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25525266

RESUMO

SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to (8-9)FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing (8-9)FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind (2-5)FNI in addition to (8-9)FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow koff. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction.


Assuntos
Proteínas de Bactérias/química , Fibronectinas/química , Streptococcus equi/química , Proteínas de Bactérias/genética , Fibronectinas/genética , Estrutura Terciária de Proteína , Streptococcus equi/genética
16.
J Biol Chem ; 289(32): 22490-9, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-24962582

RESUMO

BBK32 is a fibronectin (FN)-binding protein expressed on the cell surface of Borrelia burgdorferi, the causative agent of Lyme disease. There is conflicting information about where and how BBK32 interacts with FN. We have characterized interactions of a recombinant 86-mer polypeptide, "Bbk32," comprising the unstructured FN-binding region of BBK32. Competitive enzyme-linked assays utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies showed that Bbk32 binding involves both the fibrin-binding and the gelatin-binding domains of the 70-kDa N-terminal region (FN70K). Crystallographic and NMR analyses of smaller Bbk32 peptides complexed, respectively, with (2-3)FNI and (8-9)FNI, demonstrated that binding occurs by ß-strand addition. Isothermal titration calorimetry indicated that Bbk32 binds to isolated FN70K more tightly than to intact FN. In a competitive enzyme-linked binding assay, complex formation with Bbk32 enhanced binding of FN with mAbIII-10 to the (10)FNIII module. Thus, Bbk32 binds to multiple FN type 1 modules of the FN70K region by a tandem ß-zipper mechanism, and in doing so increases accessibility of FNIII modules that interact with other ligands. The similarity in the FN-binding mechanism of BBK32 and previously studied streptococcal proteins suggests that the binding and associated conformational change of FN play a role in infection.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Fibronectinas/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
17.
Invest Ophthalmol Vis Sci ; 54(13): 8337-44, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24265020

RESUMO

PURPOSE: Fibrillins are the major constituent of tissue microfibrils, which form the ocular zonule. In Marfan syndrome (MFS), FBN1 mutations lead to ectopia lentis. The goal of this work was to investigate zonule composition and formation in fibrillin-deficient and wild-type mice. METHODS: Immunofluorescence staining of eyes from wild-type, Fbn1-deficient, and Fbn2-deficient mice, as well as other species, was performed using monospecific fibrillin 1 and fibrillin 2 antibodies. The zonule of Fbn1-deficient and Fbn2-deficient mice was studied by electron microscopy. Microfibril formation in vitro was evaluated by immunofluorescence microscopy of cultured nonpigmented ciliary epithelial cells and fibroblasts. RESULTS: A zonule was present in both Fbn1-deficient and Fbn2-deficient mouse eyes. Immunofluorescence demonstrated that the zonule of Fbn1-deficient mice, wild-type mice, rats, and hamsters contained fibrillin 2. The zonule of Fbn2(-/-) mice contained fibrillin 1. Fibrillin 1 and fibrillin 2 colocalized in microfibrils formed in human nonpigmented ciliary epithelium cultures. Like fibrillin 1, fibrillin 2 microfibril assembly was fibronectin dependent and initiated by cell surface punctate deposits that elongated to form microfibrils. CONCLUSIONS: These data suggest that fibrillin 1 assembly and fibrillin 2 assembly share similar mechanisms. Microfibril composition depends substantially on the local levels of fibrillin isoforms and is not highly selective in regard to the isoform. This raises the intriguing possibility that the zonule could be strengthened in MFS by inducing fibrillin 2 expression in ciliary epithelium. The presence of fibrillin 2 in the murine zonule and an intact zonule in Fbn1-knockout mice may limit the utility of rodent models for studying ectopia lentis in MFS.


Assuntos
Corpo Ciliar/metabolismo , Cristalino/metabolismo , Ligamentos/metabolismo , Síndrome de Marfan/prevenção & controle , Proteínas dos Microfilamentos/metabolismo , Idoso , Animais , Bovinos , Células Cultivadas , Corpo Ciliar/citologia , Cricetinae , Ectopia do Cristalino/metabolismo , Ectopia do Cristalino/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Terapia Genética , Humanos , Ligamentos/ultraestrutura , Síndrome de Marfan/metabolismo , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley
18.
Matrix Biol ; 31(6): 360-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22986508

RESUMO

Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. The control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we developed a microtiter assay of FN deposition by human fibroblasts. The assay provides a robust read-out of FN assembly. Alexa 488-FN (A488-FN) was added to cell monolayers, and the total fluorescence intensity of deposited A488-FN was quantified. The fluorescence intensity of deposited A488-FN correlated with the presence of FN fibrils visualized by fluorescence microscopy. The assay Z' values were 0.67 or 0.54, respectively, when using background values of fluorescence either with no added A488-FN or with A488-FN added together with a known inhibitor of FN deposition. The assay was used to screen libraries comprising 4160 known bioactive compounds. Nine compounds were identified as non- or low-cytotoxic inhibitors of FN assembly. Four (ML-9, HA-100, tyrphostin and imatinib mesylate) are kinase inhibitors, a category of compounds known to inhibit FN assembly; two (piperlongumine and cantharidin) are promoters of cancer cell apoptosis; and three (maprotiline, CGS12066B, and aposcopolamine) are modulators of biogenic amine signaling. The latter six compounds have not been recognized heretofore as affecting FN assembly. The assay is straight-forward, adapts to 96- and 384-well formats, and should be useful for routine measurement of FN deposition and HTS. Screening of more diverse chemical libraries and identification of specific and efficient modulators of FN fibrillogenesis may result in therapeutics to control excessive connective tissue deposition.


Assuntos
Fibronectinas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Titulometria
19.
PLoS One ; 7(3): e32762, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22403705

RESUMO

Thrombospondin-1 (TSP-1) is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-ß-N-acetylglucosaminylation. Previously, O-ß-N-acetylglucosamine (O-ß-GlcNAc) was found on a threonine in the loop between the fifth and sixth cysteines of the 20(th) epidermal growth factor (EGF)-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with ß-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da) and xylose (+132 Da) separately from modification with N-acetyl hexosamine (+203 Da). Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence (575)CPPGYSGNGIQC(586). These results demonstrate that O-ß-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.


Assuntos
Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fator de Crescimento Epidérmico/química , Espaço Extracelular/metabolismo , Oxigênio , Trombospondina 1/química , Trombospondina 1/metabolismo , Sequência de Aminoácidos , Animais , Sequência Consenso , Drosophila melanogaster , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA
20.
J Biol Chem ; 287(16): 13303-12, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22351755

RESUMO

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel ß-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by ß-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit ß-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.


Assuntos
Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Adesinas Bacterianas/metabolismo , Regulação Alostérica/fisiologia , Animais , Anticorpos Monoclonais/metabolismo , Ligação Competitiva/fisiologia , Mapeamento de Epitopos , Fibrina/metabolismo , Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Ligantes , Camundongos , Estrutura Terciária de Proteína , Solubilidade , Streptococcus pyogenes/metabolismo
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