Modeling integrin and plasma-polymerized pyrrole interactions: chemical diversity relevance for cell regeneration.

Protein-engineered biomaterials represent a powerful approach to increase biofunctional activity like tissue repair and celular proliferation. Among these materials, integrins and the development of their specific interactions with plasma-polymerized pyrrole (PPPy) are promising biomaterial for tissue regeneration. In this paper, we studied the molecular recognition in the active site of three integrins (α5ß1, αvß3 and αIIbß3) with PPPy using the structure proposed by Kumar et al. PPPy molecule has three sites to incorporate different species, we worked mainly with the functional groups, -NH2 and -OH groups according to our IR spectroscopic results. We carried out docking studies to find the better conformational couplings and to determine electrostatic (ΔGelec) and non-electrostatic (ΔGnon-elec) contributions to the binding free energy (ΔGb) of these complexes we used Adaptive Poisson-Bolztmann program (APBS). Our results indicated that when incorporating -1H-azirine, -NH2 or -OH group in PPPy structure, interactions with integrins were favorable, as indicated by correspondent ΔGb values. These interactions were mainly triggered by Coulomb interactions, an important term in the electrostatic component. Furthermore, our studies suggest that some residues of integrins α5ß1, αvß3 and αIIbß3 like aspartates are important for the binding to PPPy structures. Detailed interactions between integrin α5ß1 and PPPy structures were revealed by molecular dynamics simulations. We used this particular integrin structure because of its favorable ΔGb as well as its major cellular receptor for the extracellular matrix protein fibronectin. Clustering analysis allowed us to carry out focused docking studies and to determine the time evolution of the ΔGb values. By incorporating -NH2 into PPPy structure, ΔGb values were very favorable during the course of the dynamics simulations by the establishment of hydrogen bonds with Asn224 and/orAsp227 residues, which are part of the integrin α5ß1 pocket. However, for the integrin α5ß1-PPPy-1H-azirine complex and the rest of the functional groups, the ΔGb values were less favorable, although PPPy was found at a distance of less than 5 Å from the active site residues. This work is complementary to the previous studies made employing PPPy nanoparticles for a variety of tissue engineering applications, and were done to enlighten the role played by the amino group of the PPPy in its integrin recognition process.

SPARC Controls Melanoma Cell Plasticity through Rac1.

Cell transition to a more aggressive mesenchymal-like phenotype is a hallmark of cancer progression that involves different steps and requires tightly regulated cell plasticity. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein that promotes this transition in various malignant cell types, including melanoma cells. We found that suppression of SPARC expression in human melanoma cells compromised cell migration, adhesion, cytoskeleton structure, and cell size. These changes involved the Akt/mTOR pathway. Re-expression of SPARC or protein addition restored all the cell features. Suppression of SPARC expression was associated with increased Rac1-GTP levels and its membrane localization. Expression of the dominant negative mutant of Rac1 counteracted almost all the changes observed in SPARC-deficient cells. Overall, these data suggest that most of the SPARC-mediated effects occurred mainly through the blockade of Rac1 activity.

Adhesion and proliferation of HeLa and fibroblast cells on chemically-modified gold surfaces.

The development of materials that allow proper functioning of cells on solid supports is directly relevant to the construction of living-cell biosensors. Both physical and chemical properties of the surfaces have been shown to be critical in this field. Our aim is to report correlations between chemical properties of surfaces and cell behavior by studying adhesion, viability and proliferation of fibroblasts and HeLa cells. Neither fibroblasts nor HeLa cells adhered to a hydrophobic surface. Fibroblasts were able to attach and proliferate well on all other surfaces tested. In contrast, on some surfaces where HeLa cells adhered and were viable, proliferation decreased by half while on others proliferation was not affected. Proliferation was significantly correlated with the level of adsorption of serum proteins on the surface (quantified by surface plasmon resonance), but not with surface wettability (water contact angle). Interestingly, surfaces modified with COOH and HSO3 groups were the ones that favored most protein adsorption and allowed the best measures for HeLa cell proliferation. The decrease of HeLa cell proliferation on surfaces covered with poly-L-lysine (PL) was related with the profile of integrin expression. Compared to a polystyrene control surface, there was an increase in αV and αVß3 and a decrease in α2 and α3, indicating that migration rather than proliferation could be favored on PL functionalized surfaces. These results indicate that charge is more important than wettability to determine biocompatibility.

Jararhagin, a hemorrhagic snake venom metalloproteinase from Bothrops jararaca.

Jararhagin is a metalloproteinase isolated from Bothrops jararaca snake venom, which has been extensively studied. These studies showed its involvement on most of the systemic and local damaging effects of snakebite envenomings. In this review we comment on the major targets of jararhagin as the vascular endothelium, platelets and coagulation factors and also its action on other cell systems as inflammatory cells and their mediators, cancer and cell signaling. The mechanisms of jararhagin action are discussed together with structural features essential for the expression of its biological activities. The studies reviewed here denote jararhagin as a prototype for studies of snake venom metalloproteinases, bringing new insights into cellular-matrix interactions and adding for the improvement of snakebite treatment.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.

It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.

[Integrins: a family of cell adhesion receptors]. / Las integrinas: una familia de receptores de adhesión celular.

Integrins are a family of cell adhesion molecules (CAM's) that mediate the communication between the intracellular and the extracellular compartments. The growing interest in CAM's is due to the essential role they play in cell-cell and cell-matrix recognition processes. These receptors are formed by a non-covalently associated glycoprotein complex of two distinct polypeptide chains, called, alpha and beta. The association of different subunits results in the formation of, at least, 16 different integrins that provide cells with a great versatility in their adhesion properties. An integrin molecule comprises a cytoplasmic domain that interacts with the cytoskeleton, a transmembranous domain and an extracellular domain that binds to one or more ligands. beta 1, beta 2 and beta 3, are the best characterized integrin subfamilies; they are expressed, in different amounts, in epithelial and endothelial cells, leukocytes, fibroblasts and platelets. b1 integrins are essentially involved in cell-extracellular matrix interactions and beta 2 subfamily in leukocyte-leukocyte and leukocyte-endothelial cell communications. The integrin subfamily beta 3 mediates the adhesion of platelets with fibrinogen and other ligands. During embryonic development, integrins in association with other CAM's, play an essential role in cell migration and morphogenesis. Moreover, in processes like inflammation, wound healing and thrombosis, integrins and other CAM's mediate the interactions among the injured tissue and circulating cells. In two genetic diseases like the leukocyte adhesion deficiency and the Glanzmann's thrombasthenia an impairment in leukocyte-endothelial cell interactions and platelet aggregation is detected, due to deficiencies or abnormalities in beta 2 or beta 3 integrin subfamilies, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Las integrinas: una familia de receptores de adhesión celular / Integrins: a family of cell adhesion receptors

Las integrinas constituyen una familia de moléculas de adhesión (CAMP's) que integran el compartimiento intracelular con el extracelular. El interés creciente en las mismas reside en el papel que juegan en los procesos de reconocimiento célula/célula y célula/sustrato. Estos receptores son glicoproteinas formadas por un heterodímero con 2 subunidades polipeptídicas (alfa y ß) unidas en forma no covalente. La asociación de distintas subunidades permite la formación de, por lo menos, 16 integrinas lo que provee a la célula de una amplia versatilidad en sus propiedades de adhesión. Estas moléculas poseen un dominio citoplasmático con el cual interactúan con el citoesqueleto, un dominio transmembranoso y un dominio extracelular que se une a uno o más ligandos. Las integrinas de las subfamilias ß1, ß2 y ß3 que han sido las más estudiadas, se expresan, en distinta proporción en células epiteliais y endoteliales, leucocitos, fibroblastos y plaquetas. Aquellas pertenecientes a la subfamilia ß1 median, fundamentalmente, la adhesión de la célula a las proteínas de la matriz extracelular mientras que las correspondientes a la subfamilia ß2 intervienen en la interacción leucocito/leucocito y leucocito/célula endotelial. La subfamilia ß3 actúa como mediadora en la adhesión de las plaquetas con el fibrinógeno y otros ligandos. Durante el desarrollo embrionario, las integrinas en asociación con otras CAM's, constituyen uno de los factores esenciales en la migración celular y morfogénesis. Por otra parte, en fenómenos como la inflamación, la cicatrización y la trombosis las integrinas permiten la interacción entre el tejido dañado y las células circulantes. En enfermedades genéticas como el síndrome de deficiencia de adhesión leucocitaria o la trombastenia de Glanzmann se detectan alteraciones en la adhesión leucocito/endotelio o en la agregación plaquetaria debidas a deficiencias en las integrinas de las subfamilias ß2 o ß3, respectivamente. En la invasión tumoral y metástasis las integrinas están involucradas en feómenos tales como el desprendimiento de las células de la matriz extracelular, la migración y la colonización tumoral. Las integrinas de las subfamilias ß1 y ß3 median la asociación de las plaquetas con las células tumorales facilitando el pasaje de estas últimas a través del endotelio. El uso de anticuerpos específicos contra integrinas o formas solubles de estos receptores o de sus ligandos podría ser de utilidad terapéutica al modular los fenómenos de inflamación, trombosis o invasión tumoral

Las integrinas: una familia de receptores de adhesión celular / Integrins: a family of cell adhesion receptors

Las integrinas constituyen una familia de moléculas de adhesión (CAMPs) que integran el compartimiento intracelular con el extracelular. El interés creciente en las mismas reside en el papel que juegan en los procesos de reconocimiento célula/célula y célula/sustrato. Estos receptores son glicoproteinas formadas por un heterodímero con 2 subunidades polipeptídicas (alfa y ß) unidas en forma no covalente. La asociación de distintas subunidades permite la formación de, por lo menos, 16 integrinas lo que provee a la célula de una amplia versatilidad en sus propiedades de adhesión. Estas moléculas poseen un dominio citoplasmático con el cual interactúan con el citoesqueleto, un dominio transmembranoso y un dominio extracelular que se une a uno o más ligandos. Las integrinas de las subfamilias ß1, ß2 y ß3 que han sido las más estudiadas, se expresan, en distinta proporción en células epiteliais y endoteliales, leucocitos, fibroblastos y plaquetas. Aquellas pertenecientes a la subfamilia ß1 median, fundamentalmente, la adhesión de la célula a las proteínas de la matriz extracelular mientras que las correspondientes a la subfamilia ß2 intervienen en la interacción leucocito/leucocito y leucocito/célula endotelial. La subfamilia ß3 actúa como mediadora en la adhesión de las plaquetas con el fibrinógeno y otros ligandos. Durante el desarrollo embrionario, las integrinas en asociación con otras CAMs, constituyen uno de los factores esenciales en la migración celular y morfogénesis. Por otra parte, en fenómenos como la inflamación, la cicatrización y la trombosis las integrinas permiten la interacción entre el tejido dañado y las células circulantes. En enfermedades genéticas como el síndrome de deficiencia de adhesión leucocitaria o la trombastenia de Glanzmann se detectan alteraciones en la adhesión leucocito/endotelio o en la agregación plaquetaria debidas a deficiencias en las integrinas de las subfamilias ß2 o ß3, respectivamente. En la invasión tumoral y metástasis las integrinas están involucradas en feómenos tales como el desprendimiento de las células de la matriz extracelular, la migración y la colonización tumoral. Las integrinas de las subfamilias ß1 y ß3 median la asociación de las plaquetas con las células tumorales facilitando el pasaje de estas últimas a través del endotelio. El uso de anticuerpos específicos contra integrinas o formas solubles de estos receptores o de sus ligandos podría ser de utilidad terapéutica al modular los fenómenos de inflamación, trombosis o invasión tumoral (AU)