RESUMEN
Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Antígenos Thy-1/metabolismo , Astrocitos/metabolismo , Adhesión Celular , Comunicación Celular , Movimiento Celular/fisiología , Células Cultivadas , Células HEK293 , Humanos , Integrina alfa5/metabolismo , Integrina alfaVbeta3/química , Integrina alfaVbeta3/fisiología , Cinética , Neuronas/metabolismo , Transducción de Señal , Imagen Individual de Molécula/métodos , Termodinámica , Antígenos Thy-1/química , Antígenos Thy-1/fisiologíaRESUMEN
In the present study, a homologous rotavirus, ECwt, infecting small intestinal villi isolated from ICR and BALB/c mice were used as a model for identifying cell-surface molecules involved in rotavirus entry. Small-intestinal villi were treated with anti-Hsc70, anti-PDI, anti-integrin ß3 or anti-ERp57 antibodies or their corresponding F(ab')2 fragments before inoculation with rotavirus ECwt, RRV or Wa. Pretreatment of villi decreased virus infectivity by about 50-100 % depending of the rotavirus strain, antibody structure and detection assay used. Similar results were obtained by treating viral inocula with purified proteins Hsc70, PDI or integrin ß3 before inoculation of untreated villi. Rotavirus infection of villi proved to be sensitive to membrane-impermeant thiol/disulfide inhibitors such as DTNB and bacitracin, suggesting the involvement of a redox reaction in infection. The present results suggest that PDI, Hsc70 and integrin ß3 are used by both homologous and heterologous rotaviruses during infection of isolated mouse villi.
Asunto(s)
Proteínas del Choque Térmico HSC70/fisiología , Integrina alfaVbeta3/fisiología , Intestino Delgado/virología , Proteína Disulfuro Isomerasas/fisiología , Infecciones por Rotavirus/virología , Rotavirus/fisiología , Internalización del Virus , Animales , Animales Lactantes/virología , Anticuerpos/inmunología , Supervivencia Celular , Femenino , Proteínas del Choque Térmico HSC70/inmunología , Proteínas del Choque Térmico HSC70/metabolismo , Integrina alfaVbeta3/inmunología , Integrina alfaVbeta3/metabolismo , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Proteína Disulfuro Isomerasas/metabolismo , Rotavirus/aislamiento & purificación , Infecciones por Rotavirus/metabolismoRESUMEN
To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val(79) to Thr(346) (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. (99m)Technecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Fragmentos de Péptidos/farmacología , Plasminógeno/farmacología , Secuencia de Aminoácidos , Animales , Glicosilación , Humanos , Integrina alfaVbeta3/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Plasminógeno/química , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Several cell surface molecules have been implicated in rotavirus cell entry, however, their individual relevance during this process is unknown. In this work, the expression of integrins alpha2, beta2, and alpha v beta 3, the heat shock cognate protein 70, and of ganglioside GM1 in different cell lines of human and simian origin was correlated with the infectivity of four rotavirus strains. We observed that different combinations of receptor expression correlated with the infectivity of rotavirus strains, suggesting that the participation of several receptors is important for rotavirus infection. To characterize the relevance of integrins alpha2 and alpha v beta 3 in more detail, their expression was silenced using RNA interference. About 80% decrease in the cell content of integrins resulted in 15-30% decrease of infectivity of strains RRV and Wa when measured by a focus-forming assay, while there was no decrease of infectivity when measured by flow cytometry in integrin-deficient cells. Altogether these data suggest that integrins alpha2 and alpha v beta 3 do not play a major role in the rotavirus entry process.
Asunto(s)
Integrina alfa2/fisiología , Integrina beta3/fisiología , Receptores Virales/fisiología , Rotavirus/fisiología , Internalización del Virus , Animales , Línea Celular , Gangliósido G(M1)/genética , Gangliósido G(M1)/fisiología , Técnicas de Silenciamiento del Gen/métodos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Integrina alfa2/genética , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/fisiología , Integrina beta3/genética , Macaca mulatta , Receptores Virales/antagonistas & inhibidores , Receptores Virales/genéticaRESUMEN
The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation.