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1.
J Biol Chem ; 295(36): 12822-12839, 2020 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-32111735

RESUMEN

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Tetraspaninas/metabolismo , Células A549 , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Células HEK293 , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Tetraspaninas/genética
2.
Haematologica ; 104(9): 1892-1905, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-30573509

RESUMEN

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.


Asunto(s)
Calcio/metabolismo , Daño por Reperfusión Miocárdica/genética , Proteína ORAI1/genética , Tetraspaninas/genética , Trombosis de la Vena/genética , Factor de von Willebrand/genética , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Pollos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Histamina/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Transporte Iónico/efectos de los fármacos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1/metabolismo , Transducción de Señal , Tetraspaninas/metabolismo , Trombina/farmacología , Trombosis de la Vena/metabolismo , Trombosis de la Vena/patología , Factor de von Willebrand/metabolismo
3.
FASEB J ; 32(7): 3560-3573, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29430990

RESUMEN

The transmembrane protein, ADAM10 (a disintegrin and metalloprotease 10), has key physiologic functions-for example, during embryonic development and in the brain. During transit through the secretory pathway, immature ADAM10 (proADAM10) is converted into its proteolytically active, mature form (mADAM10). Increasing or decreasing the abundance and/or activity of mADAM10 is considered to be a therapeutic approach for the treatment of such diseases as Alzheimer's disease and cancer. Yet biochemical detection and characterization of mADAM10 has been difficult. In contrast, proADAM10 is readily detected-for example, in immunoblots-which suggests that mADAM10 is only a fraction of total cellular ADAM10. Here, we demonstrate that mADAM10, but not proADAM10, unexpectedly undergoes rapid, time-dependent degradation upon biochemical cell lysis in different cell lines and in primary neurons, which prevents the detection of the majority of mADAM10 in immunoblots. This degradation required the catalytic activity of ADAM10, was efficiently prevented by adding active site inhibitors to the lysis buffer, and did not affect proADAM10, which suggests that ADAM10 degradation occurred in an intramolecular and autoproteolytic manner. Inhibition of postlysis autoproteolysis demonstrated efficient cellular ADAM10 maturation with higher levels of mADAM10 than proADAM10. Moreover, a cycloheximide chase experiment revealed that mADAM10 is a long-lived protein with a half-life of approximately 12 h. In summary, our study demonstrates that mADAM10 autoproteolysis must be blocked to allow for the proper detection of mADAM10, which is essential for the correct interpretation of biochemical and cellular studies of ADAM10.-Brummer, T., Pigoni, M., Rossello, A., Wang, H., Noy, P. J., Tomlinson, M. G., Blobel, C. P., Lichtenthaler, S. F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.


Asunto(s)
Proteína ADAM10/metabolismo , Proteolisis , Animales , Línea Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 38(2): 344-352, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29146750

RESUMEN

OBJECTIVE: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. APPROACH AND RESULTS: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7R/R ) causes a marked signaling defect in platelets. Trpm7R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and ß3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. CONCLUSIONS: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.


Asunto(s)
Arteriopatías Oclusivas/sangre , Plaquetas/metabolismo , Señalización del Calcio , Calcio/sangre , Infarto de la Arteria Cerebral Media/sangre , Canales Catiónicos TRPM/sangre , Trombosis/sangre , Animales , Arteriopatías Oclusivas/genética , Arteriopatías Oclusivas/patología , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Lectinas Tipo C/sangre , Ratones Mutantes , Fosfatidilinositol 4,5-Difosfato/sangre , Fosfolipasa C beta/sangre , Fosfolipasa C gamma/sangre , Fosforilación , Glicoproteínas de Membrana Plaquetaria/metabolismo , Mutación Puntual , Receptores Proteinasa-Activados/sangre , Molécula de Interacción Estromal 1/sangre , Sinaptofisina/sangre , Canales Catiónicos TRPM/deficiencia , Canales Catiónicos TRPM/genética , Trombosis/genética , Trombosis/patología
5.
J Immunol ; 199(2): 666-676, 2017 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-28600292

RESUMEN

The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antígenos CD/genética , Cadherinas/genética , Proteínas de la Membrana/metabolismo , Linfocitos T/fisiología , Tetraspaninas/metabolismo , Migración Transendotelial y Transepitelial , Proteína ADAM10/deficiencia , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Antígenos CD/metabolismo , Linfocitos B/inmunología , Linfocitos B/fisiología , Cadherinas/metabolismo , Células Cultivadas , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/inmunología , Quimiocina CXCL16 , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Células Endoteliales/inmunología , Células Endoteliales/fisiología , Humanos , Inflamación/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Neutrófilos/inmunología , Neutrófilos/fisiología , Receptores Depuradores/genética , Receptores Depuradores/inmunología , Linfocitos T/inmunología , Tetraspaninas/genética , Tetraspaninas/inmunología
6.
J Biol Chem ; 291(7): 3145-57, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26668317

RESUMEN

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.


Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Tetraspaninas/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Plaquetas/citología , Línea Celular , Membrana Celular/enzimología , Células Cultivadas , Endotelio Vascular/citología , Activación Enzimática , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Propiedades de Superficie , Tetraspanina 29/química , Tetraspanina 29/genética , Tetraspanina 29/metabolismo , Tetraspaninas/química , Tetraspaninas/genética
7.
Biochem Soc Trans ; 45(3): 719-730, 2017 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-28620033

RESUMEN

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane protein which is essential for embryonic development through activation of Notch proteins. ADAM10 regulates over 40 other transmembrane proteins and acts as a 'molecular scissor' by removing their extracellular regions. ADAM10 is also a receptor for α-toxin, a major virulence factor of Staphylococcus aureus Owing to the importance of its substrates, ADAM10 is a potential therapeutic target for cancer, neurodegenerative diseases such as Alzheimer's and prion diseases, bacterial infection and inflammatory diseases such as heart attack, stroke and asthma. However, targetting ADAM10 is likely to result in toxic side effects. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals which interact with and regulate specific partner proteins within membrane nanodomains. Tetraspanins appear to have a cone-shaped structure with a cholesterol-binding cavity, which may enable tetraspanins to undergo cholesterol-regulated conformational change. An emerging paradigm for tetraspanin function is the regulation of ADAM10 by the TspanC8 subgroup of tetraspanins, namely Tspan5, 10, 14, 15, 17 and 33. This review will describe how TspanC8s are required for ADAM10 trafficking from the endoplasmic reticulum and its enzymatic maturation. Moreover, different TspanC8s localise ADAM10 to different subcellular localisations and may cause ADAM10 to adopt distinct conformations and cleavage of distinct substrates. We propose that ADAM10 should now be regarded as six different scissor proteins depending on the interacting TspanC8. Therapeutic targetting of specific TspanC8/ADAM10 complexes could allow ADAM10 targetting in a cell type- or substrate-specific manner, to treat certain diseases while minimising toxicity.


Asunto(s)
Proteína ADAM10/metabolismo , Tetraspaninas/metabolismo , Animales , Humanos , Conformación Proteica , Transporte de Proteínas , Especificidad por Sustrato
8.
FASEB J ; 30(6): 2311-23, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26939791

RESUMEN

C-type lectin family 14, member A (CLEC14A), is a single-pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid-like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid-like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or -3. Site-directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2-fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3-fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.-Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Endopeptidasas/metabolismo , Células Endoteliales/fisiología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica/fisiología , Serina Proteasas/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biomecánicos , Moléculas de Adhesión Celular/genética , Movimiento Celular/fisiología , Endopeptidasas/genética , Regulación de la Expresión Génica/fisiología , Humanos , Lectinas Tipo C/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Dominios Proteicos , Serina Endopeptidasas , Serina Proteasas/genética
9.
Platelets ; 28(4): 333-341, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27256961

RESUMEN

A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are ubiquitous transmembrane "molecular scissors" which proteolytically cleave, or shed, the extracellular regions of other transmembrane proteins. ADAM10 is essential for development because it cleaves Notch proteins to induce Notch signaling and regulate cell fate decisions. ADAM17 is regarded as a first line of defense against injury and infection, by releasing tumor necrosis factor α (TNFα) to promote inflammation and epidermal growth factor (EGF) receptor ligands to maintain epidermal barrier function. However, the regulation of ADAM10 and ADAM17 trafficking and activation are not fully understood. This review will describe how the TspanC8 subgroup of tetraspanins (Tspan5, 10, 14, 15, 17, and 33) and the iRhom subgroup of protease-inactive rhomboids (iRhom1 and 2) have emerged as important regulators of ADAM10 and ADAM17, respectively. In particular, they are required for the enzymatic maturation and trafficking to the cell surface of the ADAMs, and there is evidence that different TspanC8s and iRhoms target the ADAMs to distinct substrates. The TspanC8s and iRhoms have not been studied functionally on platelets. On these cells, ADAM10 is the principal sheddase for the platelet collagen receptor GPVI, and the regulatory TspanC8s are Tspan14, 15, and 33, as determined from proteomic data. Platelet ADAM17 is the sheddase for the von Willebrand factor (vWF) receptor GPIb, and iRhom2 is the only iRhom that is expressed. Induced shedding of either GPVI or GPIb has therapeutic potential, since inhibition of either receptor is regarded as a promising anti-thrombotic therapy. Targeting of Tspan14, 15, or 33 to activate platelet ADAM10, or iRhom2 to activate ADAM17, may enable such an approach to be realized, without the toxic side effects of activating the ADAMs on every cell in the body.


Asunto(s)
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Desintegrinas/metabolismo , Tetraspaninas/metabolismo , Animales , Humanos
10.
Platelets ; 28(7): 629-642, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28032533

RESUMEN

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.


Asunto(s)
Plaquetas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/genética , Tetraspaninas/genética , Adenosina Difosfato/farmacología , Animales , Ácido Araquidónico/farmacología , Plaquetas/patología , Proteínas Portadoras/farmacología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/farmacología , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Cultivo Primario de Células , Unión Proteica , Transporte de Proteínas , Transducción de Señal , Tetraspaninas/química , Tetraspaninas/deficiencia
11.
J Cell Sci ; 127(Pt 14): 3039-51, 2014 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-24928894

RESUMEN

RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, ß-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or ß-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Adhesiones Focales/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Movimiento Celular/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/metabolismo , Transducción de Señal
12.
Methods Mol Biol ; 1591: 155-168, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28349481

RESUMEN

A major impediment when studying primary human endothelial cell function is the resistance of these cells to gene transfer. Here we describe methods for transferring genes into primary endothelial cells prior to incorporation into a static adhesion assay to analyze the adhesion and migration of isolated lymphocytes. Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) cells are initially transfected with plasmids containing the lentiviral packaging and envelope genes and the target sequence, such as a gene of interest or short hairpin loop RNA (shRNA). These cells produce lentivirus packaged with this target sequence and are used to transduce primary human umbilical vein endothelial cells (HUVECs). Human peripheral blood lymphocytes (PBLs) isolated from venous blood are co-incubated with lentivirally transduced cytokine-stimulated endothelial cells to assess lymphocyte adhesion in a static adhesion assay. Direct observations of lymphocyte adhesion and migration over a time course can also be made. In general, lentiviral transduction of primary endothelial cells provides an invaluable system to manipulate gene expression levels when studying the cellular adhesion dynamics that regulate leukocyte adhesion and extravasation.


Asunto(s)
Células Endoteliales/fisiología , Vectores Genéticos/genética , Lentivirus/genética , Linfocitos/fisiología , Adhesión Celular/genética , Línea Celular , Células Endoteliales/virología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Linfocitos/virología , Plásmidos/genética , ARN Interferente Pequeño/genética , Transducción Genética/métodos , Transfección/métodos
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