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1.
J Biol Chem ; 298(9): 102323, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35931112

RESUMEN

Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.


Asunto(s)
Fibronectinas , Integrina alfa5beta1 , Dominios y Motivos de Interacción de Proteínas , Sitios de Unión , Adhesión Celular , Medición de Intercambio de Deuterio , Fibronectinas/química , Fibronectinas/genética , Integrina alfa5beta1/química , Mutación , Oligopéptidos/química , Solventes
2.
Matrix Biol ; 96: 87-103, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33157226

RESUMEN

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5ß1 integrins in endothelial cells in vitro, and α5ß1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5ß1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5ß1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5ß1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.


Asunto(s)
Aterosclerosis/metabolismo , Células Endoteliales/citología , Integrina alfa5beta1/metabolismo , Talina/genética , Animales , Aterosclerosis/genética , Adhesión Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Humanos , Integrina alfa5beta1/química , Ratones , Mutación , FN-kappa B/metabolismo , Conformación Proteica , Transducción de Señal
3.
Chembiochem ; 22(7): 1151-1160, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33140906

RESUMEN

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists' efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvß3 and α5ß1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.


Asunto(s)
Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Neoplasias/patología , Oligopéptidos/metabolismo , Animales , Humanos , Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Ligandos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Oligopéptidos/química , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Unión Proteica
4.
Micron ; 137: 102888, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32554186

RESUMEN

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.


Asunto(s)
Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Imagen Individual de Molécula/métodos , Sindecano-4/química , Sindecano-4/metabolismo , Adhesión Celular , Línea Celular Tumoral , Matriz Extracelular , Humanos , Integrina alfa5beta1/genética , Sindecano-4/genética , Neoplasias de la Vejiga Urinaria , Vitronectina/metabolismo
5.
Sci Rep ; 10(1): 7410, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-32366988

RESUMEN

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.


Asunto(s)
Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Isoxazoles/química , Oligopéptidos/química , Peptidomiméticos , Animales , Bovinos , Adhesión Celular , Fibronectinas/química , Proteínas Fluorescentes Verdes/química , Humanos , Concentración de Iones de Hidrógeno , Células K562 , Ligandos , Sistema de Señalización de MAP Quinasas , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular
6.
Sci Rep ; 10(1): 5795, 2020 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-32242141

RESUMEN

Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor and has been described for G protein-coupled receptors. However, it has not yet been described for ligands interacting with integrins without αI domain. Here, we show by molecular dynamics simulations that four side chain-modified derivatives of tauroursodeoxycholic acid (TUDC), an agonist of α5ß1 integrin, differentially shift the conformational equilibrium of α5ß1 integrin towards the active state, in line with the extent of ß1 integrin activation from immunostaining. Unlike TUDC, 24-nor-ursodeoxycholic acid (norUDCA)-induced ß1 integrin activation triggered only transient activation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase and, consequently, only transient insertion of the bile acid transporter Bsep into the canalicular membrane, and did not involve activation of epidermal growth factor receptor. These results provide evidence that TUDC and norUDCA exert a functional selectivity at α5ß1 integrin and may provide a rationale for differential therapeutic use of UDCA and norUDCA.


Asunto(s)
Colagogos y Coleréticos/farmacología , Integrina alfa5beta1/metabolismo , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas , Ácido Tauroquenodesoxicólico/farmacología , Ácido Ursodesoxicólico/farmacología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Animales , Sitios de Unión , Colagogos y Coleréticos/química , Receptores ErbB/metabolismo , Integrina alfa5beta1/química , Hígado/efectos de los fármacos , Masculino , Simulación del Acoplamiento Molecular , Unión Proteica , Ratas , Ratas Wistar , Ácido Tauroquenodesoxicólico/química , Ácido Ursodesoxicólico/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
7.
Sci Rep ; 10(1): 2580, 2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-32054952

RESUMEN

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5ß1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.


Asunto(s)
Huesos/diagnóstico por imagen , Colorantes Fluorescentes/farmacología , Imagen Molecular , Osteogénesis/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Durapatita/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Integrina alfa5beta1/química , Integrina alfa5beta1/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Oseointegración/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Pamidronato/farmacología , Tomografía Computarizada por Rayos X
8.
Nano Lett ; 20(2): 1183-1191, 2020 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-31908168

RESUMEN

Cancer cell-matrix interactions have been shown to enhance cancer cell survival via the activation of pro-survival signaling pathways. These pathways are initiated at the site of interaction, i.e., integrins, and thus, their inhibition has been the target of therapeutic strategies. Individual roles for fibronectin-binding integrin subtypes αvß3 and α5ß1 have been shown for various cellular processes; however, a systematic comparison of their function in adhesion-dependent chemoresistance is lacking. Here, we utilize integrin subtype-specific peptidomimetics for αvß3 and α5ß1, both as blocking agents on fibronectin-coated surfaces and as surface-immobilized adhesion sites, in order to parse out their role in breast cancer cell survival. Block copolymer micelle nanolithography is utilized to immobilize peptidomimetics onto highly ordered gold nanoparticle arrays with biologically relevant interparticle spacings (35, 50, or 70 nm), thereby providing a platform for ascertaining the dependence of ligand spacing in chemoprotection. We show that several cellular properties-morphology, focal adhesion formation, and migration-are intricately linked to both the integrin subtype and their nanospacing. Importantly, we show that chemotherapeutic drug sensitivity is highly dependent on both parameters, with smaller ligand spacing generally hindering survival. Furthermore, we identify ligand type-specific patterns of drug sensitivity, with enhanced chemosurvival when cells engage αvß3 vs α5ß1 on fibronectin; however, this is heavily reliant on nanoscale spacing, as the opposite is observed when ligands are spaced at 70 nm. These data imply that even nanoscale alterations in extracellular matrix properties have profound effects on cancer cell survival and can thus inform future therapies and drug testing platforms.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Adhesión Celular/genética , Integrina alfa5beta1/genética , Integrina alfaVbeta3/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Fibronectinas/química , Fibronectinas/genética , Oro/química , Humanos , Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Ligandos , Nanopartículas del Metal/química , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
9.
Biophys J ; 117(4): 688-695, 2019 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-31337547

RESUMEN

Dynamic single-molecule force spectroscopy was performed to monitor the unbinding of fibronectin with the proteoglycans syndecan-4 (SDC4) and decorin and to compare this with the unbinding characteristics of α5ß1-integrin. A single energy barrier was sufficient to describe the unbinding of both SDC4 and decorin from fibronectin, whereas two barriers were observed for the dissociation of α5ß1-integrin from fibronectin. The outer (high-affinity) barriers in the interactions of fibronectin with α5ß1-integrin and SDC4 are characterized by larger barrier heights and widths and slower dissociation rates than those of the inner (low-affinity) barriers in the interactions of fibronectin with α5ß1-integrin and decorin. These results indicate that SDC4 and (ultimately) α5ß1-integrin have the ability to withstand deformation in their interactions with fibronectin, whereas the decorin-fibronectin interaction is considerably more brittle.


Asunto(s)
Decorina/metabolismo , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Sindecano-4/metabolismo , Sitios de Unión , Decorina/química , Fibronectinas/química , Humanos , Integrina alfa5beta1/química , Unión Proteica , Sindecano-4/química , Termodinámica
10.
Methods Mol Biol ; 1998: 93-103, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31250296

RESUMEN

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5ß1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5ß1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Integrina alfa5beta1/metabolismo , Microscopía Intravital/métodos , Análisis de la Célula Individual/métodos , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Colorantes Fluorescentes/química , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Integrina alfa5beta1/química , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Imagen Molecular/métodos , Sondas Moleculares/química
12.
Food Chem ; 272: 751-757, 2019 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-30309606

RESUMEN

Marine shellfish provides a series of biofunctionality account of its high-protein level. In this study, the osteogenic effect of a novel peptide, YRGDVVPK, from Crassostrea gigas protein hydrolysates on preosteoblast MC3T3-E1 proliferation was examined. Synthetic peptide with 100 nM significantly promoted the proliferation of MC3T3-E1 cells for a treatment of 72 h assayed by MTT method, and which was confirmed by the increase of alkaline phosphatase (ALP) activity. The peptide, YRGDVVPK, was docked with integrin α5ß1 (PDB ID: 3VI4), which is a surface receptor of MC3T3-E1. The interaction of the peptide with integrin α5ß1 (PDB ID: 3VI4) was analyzed by the molecular modeling algorithm of CDOCKER, which showed a more stable combination than the original ligand. The results suggested the novel peptide could promote the preosteoblast MC3T3-E1 proliferation probably by activating the signaling pathway of MAPK, which is induced through binding with peptide YRGDVVPK.


Asunto(s)
Crassostrea/metabolismo , Péptidos/metabolismo , Tubulina (Proteína)/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Estructura Terciaria de Proteína
13.
Biomed Mater ; 13(5): 055009, 2018 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-29967311

RESUMEN

Volumetric muscle loss is debilitating and involves extensive rehabilitation. One approach to accelerate healing, rehabilitation, and muscle function is to repair damaged skeletal muscle using regenerative medicine strategies. In sports medicine and orthopedics, a common clinical approach is to treat minor to severe musculoskeletal injuries with platelet-rich plasma (PRP) injections. While these types of treatments have become commonplace, there are limited data demonstrating their effectiveness. The goal of this study was to determine the effect of PRP on myoblast gene expression and protein production when incorporated into a polymer fiber. To test this, we generated extracellular matrix mimicking scaffolds using aligned polydioxanone (PDO) fibers containing lyophilized PRP (SmartPReP® 2, Harvest Technologies Corporation, Plymouth, MA). Scaffolds with PRP caused a dose-dependent increase in myogenin and myosin heavy chain but did not affect myogenic differentiation factor-1 (MyoD). Integrin α7ß1D decreased and α5ß1A did not change in response to PRP scaffolds. ERK inhibition decreased myogenin and increased Myod on the PDO-PRP scaffolds. Taken together, these data suggest that alignment and PRP produce a substrate-dependent, ERK-dependent, and dose-dependent effect on myogenic differentiation.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Miogenina/química , Plasma Rico en Plaquetas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Matriz Extracelular , Perfilación de la Expresión Génica , Humanos , Integrina alfa5beta1/química , Integrinas/química , Ratones , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos , Proteína MioD/química , Mioblastos/citología , Cadenas Pesadas de Miosina/química , Polidioxanona/química , Polímeros/química , Medicina Regenerativa/métodos , Andamios del Tejido
14.
ACS Nano ; 12(4): 3609-3622, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29633832

RESUMEN

Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5ß1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA-Fn-integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem ß-zipper, and that the FnBPA-Fn complex further binds to immobilized α5ß1 integrins with a strength much higher than that of the classical Fn-integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites via allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial-host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Integrina alfa5beta1/metabolismo , Staphylococcus aureus/metabolismo , Estrés Mecánico , Adhesinas Bacterianas/química , Adhesión Bacteriana , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa5beta1/química , Tamaño de la Partícula , Staphylococcus aureus/citología , Propiedades de Superficie
15.
Biotechnol Adv ; 36(1): 208-227, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29155160

RESUMEN

Integrins are cell adhesion receptors predominantly important during normal and tumor angiogenesis. A sequence present on several extracellular matrix proteins composed of Arg-Gly-Asp (RGD) has attracted attention due to its role in cell adhesion mediated by integrins. The development of ligands that can bind to integrins involved in tumor angiogenesis and brake disease progression has resulted in new investigational drug entities reaching the clinical trial phase in humans. The use of integrin-specific ligands can be useful for the vascularization of regenerative medicine constructs, which remains a major limitation for translation into clinical practice. In order to enhance vascularization, immobilization of integrin-specific RGD peptidomimetics within constructs is a recommended approach, due to their high specificity and selectivity towards certain desired integrins. This review endeavours to address the potential of peptidomimetic-coated biomaterials as vascular network promoters for regenerative medicine purposes. Clinical studies involving molecules tracking active integrins in cancer angiogenesis and reasons for their failure are also addressed.


Asunto(s)
Inhibidores de la Angiogénesis , Integrina alfa5beta1 , Integrina alfaVbeta3 , Neovascularización Patológica , Medicina Regenerativa , Animales , Descubrimiento de Drogas , Humanos , Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Ratones , Ingeniería de Tejidos
16.
FEBS J ; 284(23): 4143-4157, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29055076

RESUMEN

The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagYRRII ), CagY C-terminal domain (CagYB10 ) and integrin α5ß1 ectodomain (α5ß1E ) or headpiece domain (α5ß1HP ). We found that CagI, CagA, CagL and CagYB10 but not CagYRRII were able to interact with α5ß1E with affinities similar to the one observed for α5ß1E interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagYB10 affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagYB10 interacted with the α5ß1HP . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagYB10 , and CagA and CagI. Altogether, our data map the network of interactions between host-cell α5ß1 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection.


Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/metabolismo , Integrina alfa5beta1/metabolismo , Mapeo de Interacción de Proteínas/métodos , Sistemas de Secreción Tipo IV/metabolismo , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Células CHO , Cricetinae , Cricetulus , Helicobacter pylori/genética , Humanos , Integrina alfa5beta1/química , Integrina alfa5beta1/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Resonancia por Plasmón de Superficie , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/genética , Difracción de Rayos X
17.
ChemMedChem ; 12(10): 738-750, 2017 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-28403559

RESUMEN

Herein we report the design and development of α5 ß1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against αv ß3 , αv ß5 , and α5 ß1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α5 ß1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors.


Asunto(s)
Curcumina/administración & dosificación , Curcumina/farmacología , Portadores de Fármacos/química , Integrina alfa5beta1/química , Nanotubos de Carbono/química , Oligopéptidos/química , Animales , Anticuerpos Monoclonales/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcumina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/inmunología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Células 3T3 NIH , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Tamaño de la Partícula , Relación Estructura-Actividad , Células Tumorales Cultivadas
18.
EMBO J ; 36(5): 629-645, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28122868

RESUMEN

We show that the three conformational states of integrin α5ß1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5ß1 On the surface of K562 cells, α5ß1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.


Asunto(s)
Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Termodinámica
19.
PLoS One ; 11(12): e0167663, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27973577

RESUMEN

Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5ß1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5ß1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5ß1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed.


Asunto(s)
Vacunas contra el SIDA/química , Sistemas de Liberación de Medicamentos/métodos , Integrina alfa5beta1/química , Nanopartículas/química , Poliésteres/química , Vacunas contra el SIDA/inmunología , Animales , Adhesión Celular/fisiología , Línea Celular Tumoral , Femenino , Fibronectinas/química , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Estudios Prospectivos , Proteínas Recombinantes/química
20.
Sci Rep ; 6: 35954, 2016 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-27786286

RESUMEN

Mechanical force regulates a broad range of molecular interactions in biology. Three types of counterintuitive mechanical regulation of receptor-ligand dissociation have been described. Catch bonds are strengthened by constant forces, as opposed to slip bonds that are weakened by constant forces. The phenomenon that bonds become stronger with prior application of cyclic forces is termed cyclic mechanical reinforcement (CMR). Slip and catch bonds have respectively been explained by two-state models. However, they assume fast equilibration between internal states and hence are inadequate for CMR. Here we propose a three-state model for CMR where both loading and unloading regulate the transition of bonds among the short-lived, intermediate, and long-lived state. Cyclic forces favor bonds in the long-lived state, hence greatly prolonging their lifetimes. The three-state model explains the force history effect and agrees with the experimental CMR effect of integrin α5ß1-fibronectin interaction. This model helps decipher the distinctive ways by which molecular bonds are mechanically strengthened: catch bonds by constant forces and CMR by cyclic forces. The different types of mechanical regulation may enable the cell to fine tune its mechanotransduction via membrane receptors.


Asunto(s)
Mecanotransducción Celular/fisiología , Modelos Biológicos , Fenómenos Biomecánicos , Moléculas de Adhesión Celular/fisiología , Metabolismo Energético , Matriz Extracelular/fisiología , Fibronectinas/fisiología , Integrina alfa5beta1/química , Integrina alfa5beta1/fisiología , Conformación Proteica
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