RESUMEN
Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19-224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor-related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Unión Proteica , Dominios Proteicos , Alineación de Secuencia , Vía de Señalización WntRESUMEN
LRP5 and LRP6 are proteins predicted to contain four six-bladed ß-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína Wnt1/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Morfogenéticas Óseas/genética , Calorimetría , Línea Celular , Cristalografía , ADN Complementario , Marcadores Genéticos/genética , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Proteína Wnt1/genéticaRESUMEN
SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type beta 1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the beta1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 beta 1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner alpha subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type beta1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of beta 1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.
Asunto(s)
Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Integrina beta1/genética , Queratinocitos/fisiología , Mutación Puntual , Secuencia de Aminoácidos , Animales , Carcinoma de Células Escamosas , Movimiento Celular/fisiología , Embrión de Pollo , Humanos , Integrina beta1/metabolismo , Queratinocitos/citología , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5-4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.
Asunto(s)
Glicoproteínas/antagonistas & inhibidores , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/sangre , Procolágeno/sangre , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos/farmacología , Femenino , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos BALB C , Regulación hacia ArribaRESUMEN
Earlier studies proved the success of using chemically functionalised multi-walled carbon nanotubes (f-MWNTs) as nanocarriers to the brain. Little insight into the kinetics of brain distribution of f-MWNTs in vivo has been reported. This study employed a wide range of qualitative and quantitative techniques with the aim of shedding the light on f-MWNT's brain distribution following intravenous injection. γ-Scintigraphy quantified the uptake of studied radiolabelled f-MWNT in the whole brain parenchyma and capillaries while 3D-single photon emission computed tomography/computed tomography imaging and autoradiography illustrated spatial distribution within various brain regions. Raman and multiphoton luminescence together with transmission electron microscopy confirmed the presence of intact f-MWNT in mouse brain, in a label-free manner. The results evidenced the presence of f-MWNT in mice brain parenchyma, in addition to brain endothelium. Such information on the rate and extent of regional and cellular brain distribution is needed before further implementation into neurological therapeutics can be made.
Asunto(s)
Encéfalo/metabolismo , Nanotubos de Carbono , Animales , Autorradiografía , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Capilares/metabolismo , Dexametasona/farmacología , Portadores de Fármacos , Endotelio/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Espectrometría Raman , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
A number of proteins have been shown to modulate canonical Wnt signalling at the cell surface, including members of the Dickkopf (Dkk) family (Baron and Rawadi in J Endocrinol 148:2635-2643, 2007; Cruciat and Niehrs in Cold Spring Harb Perspect Biol 5:a015081, 2013). The Dkk family includes four secreted proteins (Dkk1-4), which are characterised by two highly conserved cysteine-rich regions corresponding to C24-C73 and C128-C201 in human Dkk4 (hDkk4). Here we report essentially complete backbone and comprehensive side chain (15)N, (13)C and (1)H NMR assignments for full length mature hDkk4 (M1-L207) containing a short C-terminal hexa-histidine tag (E208-H222). Analysis of the backbone chemical shift data obtained indicates that there is a very limited amount of regular secondary structure, with only small stretches of ß-strand identified in both cysteine-rich regions. The N-terminal region of hDkk4 (M1-G21) and the relatively long linker between the two cysteine-rich regions (E77-Q123) appear to be unstructured and relatively mobile.
Asunto(s)
Disulfuros , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Wnt/antagonistas & inhibidores , Humanos , Estructura Secundaria de ProteínaRESUMEN
The Wnt signaling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signaling, including the control of the master transcriptional co-activator ß-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against ß-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signaling HEK293 reporter bioassay. Further characterization of one such VHH (named LL3) confirmed that it bound endogenous ß-catenin, and that it inhibited the Wnt signaling pathway downstream of the destruction complex, while production of a control Ala-substituted complementarity-determining region (CDR)3 mutant demonstrated that the inhibition of ß-catenin activity by the parent intracellular antibody was dependent on the specific CDR sequence of the antibody.
Asunto(s)
Regiones Determinantes de Complementariedad/biosíntesis , Anticuerpos de Cadena Única/biosíntesis , Vía de Señalización Wnt , beta Catenina/antagonistas & inhibidores , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/genética , Células HEK293 , Humanos , Anticuerpos de Cadena Única/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Carbon nanotubes (CNTs) exhibit unique properties which have led to their applications in the biomedical field as novel delivery systems for diagnosis and therapy purposes. We have previously reported that the degree of functionalization of CNTs is a key factor determining their biological behaviour. The present study broadens the spectrum by investigating the impact of the diameter of CNTs using two series of multi-walled CNTs (MWNTs) with distinct differences in their diameters. Both MWNTs were doubly functionalized by 1,3-dipolar cycloaddition and amidation reactions, allowing the appended functional groups to be further conjugated with radionuclide chelating moieties and antibodies or antibody fragments. All constructs possessed comparable degree of functionalization and were characterized by thermogravimetric analysis, transmission electron microscopy, gel electrophoresis and surface plasmon resonance. The MWNT conjugates were radio-labelled with indium-111, which thereby enabled in vivo single photon emission computed tomography/computed tomography (SPECT/CT) imaging and organ biodistribution study using γ-scintigraphy. The narrow MWNTs (average diameter: 9.2 nm) demonstrated enhanced tissue affinity including non-reticular endothelial tissues compared to the wider MWNTs (average diameter: 39.5 nm). The results indicate that the higher aspect ratio of narrow MWNTs may be beneficial for their future biological applications due to higher tissue accumulation.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Nanotubos de Carbono/química , Animales , Diagnóstico por Imagen/métodos , Sistemas de Liberación de Medicamentos , Ratones , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Resonancia por Plasmón de Superficie , Distribución TisularRESUMEN
In the 1990s there was a tremendous mood of optimism among pharmaceutical scientists that identification of disease-associated variations in the human genome would result in a surge of new drug targets (the 'gene-to-drug' mantra). To date the expected deluge of new drugs has not arrived. However, a small number of drugs arising directly from the study of rare human disorders showing Mendelian inheritance are now entering late stage clinical trials. Here we describe the advantages of this approach and discuss the background and early clinical trial findings with antibodies directed at a target identified in this way.
Asunto(s)
Anticuerpos/uso terapéutico , Conservadores de la Densidad Ósea/uso terapéutico , Proteínas Morfogenéticas Óseas/genética , Descubrimiento de Drogas , Marcadores Genéticos/genética , Hiperostosis/tratamiento farmacológico , Terapia Molecular Dirigida , Mutación , Osteoporosis/tratamiento farmacológico , Sindactilia/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos/farmacología , Conservadores de la Densidad Ósea/farmacología , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/inmunología , Marcadores Genéticos/inmunología , Predisposición Genética a la Enfermedad , Humanos , Hiperostosis/genética , Hiperostosis/metabolismo , Osteoporosis/genética , Osteoporosis/metabolismo , Fenotipo , Sindactilia/genética , Sindactilia/metabolismo , Resultado del TratamientoRESUMEN
The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin-neutralizing monoclonal antibody (Scl-AbIV) to gonad-intact female cynomolgus monkeys. Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl-AbIV treatment had clear anabolic effects, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle-treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group. Taken together, the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Densidad Ósea/efectos de los fármacos , Proteínas Morfogenéticas Óseas/inmunología , Huesos/metabolismo , Marcadores Genéticos/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Huesos/patología , Femenino , Macaca fascicularis , OsteogénesisRESUMEN
Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl-AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation-induced bone loss. At the end of the experiment, Scl-AbI-treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl-Ab)-treated animals. In a second experiment, treatment with Scl-AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age-matched control mice (maximum load, -26%, p < 0.05; energy, -37%, p < 0.05; ultimate strength, -33%, p < 0.05; elastic modulus, -17%, p < 0.05). A short treatment with Scl-AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl-AbI-treated animals were not significantly different from those of noncolitic age-matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclast-mediated bone resorption.
Asunto(s)
Anticuerpos/uso terapéutico , Proteínas Morfogenéticas Óseas/inmunología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/prevención & control , Colitis/complicaciones , Colitis/tratamiento farmacológico , Marcadores Genéticos/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Biomarcadores/sangre , Fenómenos Biomecánicos/efectos de los fármacos , Resorción Ósea/sangre , Resorción Ósea/complicaciones , Huesos/efectos de los fármacos , Huesos/metabolismo , Colitis/sangre , Colitis/diagnóstico por imagen , Modelos Animales de Enfermedad , Femenino , Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microtomografía por Rayos XRESUMEN
The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Osteogénesis/fisiología , Conformación Proteica , Proteínas Wnt/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Morfogenéticas Óseas/genética , Marcadores Genéticos/genética , Heparina/química , Heparina/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
FOXP3 has been identified as a key regulator of immune homeostasis. Mutations within the FOXP3 gene result in dysregulated CD4+ T-cell function and elevated cytokine production, leading to lymphoproliferative disease. FOXP3 expression in CD4+ T cells is primarily detected with the CD4+ CD25+ regulatory T-cell population. In humans the protein is detected as a doublet following immunoblot analysis. The lower band of the doublet has been identified as a splice isoform lacking a region corresponding to exon 2. The aim of this study was to investigate whether the splice variant form lacking exon 2 and a new novel splice variant lacking both exons 2 and 7, were functional inhibitors of CD4+ T-cell activation. The data generated showed that full-length FOXP3 and both splice variant forms of the protein were functional repressors of CD4+ T-cell activation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factores de Transcripción Forkhead/genética , Activación de Linfocitos/genética , Sitios de Empalme de ARN/genética , Secuencia de Bases , Antígenos CD28/inmunología , Proliferación Celular , Células Cultivadas , Clonación Molecular , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Alineación de Secuencia , TransfecciónAsunto(s)
Anticuerpos/inmunología , Desarrollo Óseo/fisiología , Proteínas Morfogenéticas Óseas/fisiología , Marcadores Genéticos/fisiología , Genoma , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos/farmacología , Desarrollo Óseo/efectos de los fármacos , Desarrollo Óseo/inmunología , Proteínas Morfogenéticas Óseas/genética , Huesos/citología , Huesos/fisiología , Marcadores Genéticos/genética , Glicoproteínas , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Hormona Paratiroidea/fisiología , Transducción de SeñalRESUMEN
It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function-associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1. These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that primes LFA-1 for ligand binding and firm adhesion.
Asunto(s)
Quimiocinas/metabolismo , Endotelio/metabolismo , Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Regulación Alostérica , Adhesión Celular , Células Cultivadas , Quimiocinas/farmacología , Citoesqueleto/metabolismo , Epítopos/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Conformación Proteica/efectos de los fármacos , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Solubilidad , Talina/metabolismoRESUMEN
E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells. A polymorphism, in which serine is substituted for arginine at position 128 (S128R) in the EGF domain, has been associated with both early-onset atherosclerosis and SLE. We investigated whether the substitution alters the ligand-binding properties of E-selectin under shear flow by studying the capacity of Chinese hamster ovary cell transfectants expressing wild type (WT) or S128R E-selectin to support interactions of neutrophils, K562 cells or HL60 cells. We initially chose to study non-fucosylated K562 cells. No interactions were observed on WT E-selectin, whereas S128R supported a transient tethering interaction of K562 cells, which was resistant to digestion with either neuraminidase or O-sialoglycoprotein endopeptidase, and, in turn, could result in firm adhesion in the presence of a beta2-integrin. HL60 cells exhibited increased rolling on S128R E-selectin. Although neuraminidase treatment inhibited all HL60 interactions with WT E-selectin, it unmasked transient tethers on S128R. We further observed that S128R recruited significantly more neutrophils than WT E-selectin, without affecting neutrophil rolling velocity. This polymorphism may therefore amplify leukocyte-endothelial cell interactions and may be a factor linking the S128R polymorphism to vascular disease.
Asunto(s)
Selectina E/metabolismo , Neuraminidasa/metabolismo , Polimorfismo Genético , Animales , Arginina , Células CHO , Cricetinae , Selectina E/genética , Células HL-60 , Humanos , Células K562 , Antígeno-1 Asociado a Función de Linfocito/genética , Metaloendopeptidasas/metabolismo , Mutagénesis Sitio-Dirigida , Células Mieloides/metabolismo , Células Mieloides/fisiología , Infiltración Neutrófila , Estimulación Física , Serina , TransfecciónRESUMEN
Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside/fisiología , Integrina alfa2/metabolismo , Integrina alfaVbeta3/metabolismo , Integrina alfaXbeta2/metabolismo , Rotavirus/fisiología , Secuencias de Aminoácidos , Proteínas de la Cápside/química , Línea Celular , Colágeno/farmacología , Fibrinógeno/farmacología , Humanos , Integrina alfa2/química , Oligopéptidos/farmacologíaRESUMEN
SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.
Asunto(s)
Antialérgicos/antagonistas & inhibidores , Ciclobutanos/síntesis química , Ciclobutanos/farmacología , Integrinas/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Aminas/química , Animales , Integrina alfa4beta1 , Integrina beta1 , Tasa de Depuración Metabólica , Ratas , Receptores de Antígeno muy Tardío , Relación Estructura-ActividadRESUMEN
SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a 1,3,5-triazine as an amide isostere are described.
Asunto(s)
Integrina alfa4beta1/antagonistas & inhibidores , Fenilalanina/química , Fenilalanina/farmacología , Triazinas/química , Triazinas/farmacología , Fenilalanina/análogos & derivados , Relación Estructura-ActividadRESUMEN
The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based VLA-4 antagonists are described.