RESUMEN
The viral agent SARS-CoV-2 clearly affects several organ systems, including the cardiovascular system. Angiopoietins are involved in vascular integrity and angiogenesis. Angiopoietin-1 (Ang1) promotes vessel stabilization, while angiopoietin-2 (Ang2), which is usually expressed at low levels, is significantly elevated in inflammatory and angiogenic conditions. Interleukin-6 (IL-6) is known to induce defective angiogenesis via the activation of the Ang2 pathway. Vasculitis and vasculopathy are some of the defining features of moderate to severe COVID-19-associated systemic disease. We investigated the serum levels of angiopoietins, as well as interleukin-6 levels and anti-SARS-CoV2 IgG titers, in hospitalized COVID-19 patients across disease severity and healthy controls. Ang2 levels were elevated in COVID-19 patients across all severity compared to healthy controls, while Ang1 levels were decreased. The patients with adverse outcomes (death and/or prolonged hospitalization) had relatively lower and stable Ang1 levels but continuously elevated Ang2 levels, while those who had no adverse outcomes had increasing levels of both Ang1 and Ang2, followed by a decrease in both. These results suggest that the dynamic levels of Ang1 and Ang2 during the clinical course may predict adverse outcomes in COVID-19 patients. Ang1 seems to play an important role in controlling Ang2-related inflammatory mechanisms in COVID-19 patients. IL-6 and anti-SARS-CoV2 spike protein IgG levels were significantly elevated in patients with severe disease. Our findings represent an informative pilot assessment into the role of the angiopoietin signaling pathway in the inflammatory response in COVID-19.
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Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor-suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the amino terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions that drive the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis.
Asunto(s)
Multimerización de Proteína , Receptor EphA2 , Proteínas Supresoras de Tumor , Humanos , Ligandos , Invasividad Neoplásica , Fosforilación , Receptor EphA2/química , Receptor EphA2/metabolismo , Transducción de Señal , Espectrometría de Fluorescencia , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.
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Neoplasias del Colon , Neoplasias Colorrectales , Masculino , Ratones , Animales , Humanos , Anticuerpos Monoclonales/farmacología , Proteínas de la Membrana/metabolismo , Proteína ADAM10/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismoRESUMEN
PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Humanos , Receptores ErbB/metabolismo , Línea Celular Tumoral , Cetuximab/farmacología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
ADAM10 is a transmembrane metalloprotease that sheds a variety of cell surface proteins, including receptors and ligands that regulate a range of developmental processes which re-emerge during tumour development. While ADAM10 is ubiquitously expressed, its activity is normally tightly regulated, but becomes deregulated in tumours. We previously reported the generation of a monoclonal antibody, 8C7, which preferentially recognises an active form of ADAM10 in human and mouse tumours. We now report our investigation of the mechanism of this specificity, and the preferential targeting of 8C7 to human tumour cell xenografts in mice. We also report the development of novel 8C7 antibody-drug conjugates that preferentially kill cells displaying the 8C7 epitope, and that can inhibit tumour growth in mice. This study provides the first demonstration that antibody-drug conjugates targeting an active conformer of ADAM10, a widely expressed transmembrane metalloprotease, enable tumour-selective targeting and inhibition.
RESUMEN
ADAM17 is upregulated in many cancers and in turn activates signaling pathways, including EGFR/ErbB, as well as those underlying resistance to targeted anti-EGFR therapies. Due to its central role in oncogenic pathways and drug resistance mechanisms, specific and efficacious monoclonal antibodies against ADAM17 could be useful for a broad patient population with solid tumors. Hence, we describe here an inhibitory anti-ADAM17 monoclonal antibody, named D8P1C1, that preferentially recognizes ADAM17 on cancer cells. D8P1C1 inhibits the catalytic activity of ADAM17 in a fluorescence-based peptide cleavage assay, as well as the proliferation of a range of cancer cell lines, including breast, ovarian, glioma, colon and the lung adenocarcinoma. In mouse models of triple-negative breast cancer and ovarian cancer, treatment with the mAb results in 78% and 45% tumor growth inhibition, respectively. Negative staining electron microscopy analysis of the ADAM17 ectodomain in complex with D8P1C1 reveals that the mAb binds the ADAM17 protease domain, consistent with its ability to inhibit the ADAM17 catalytic activity. Collectively, our results demonstrate the therapeutic potential of the D8P1C1 mAb to treat solid tumors.
RESUMEN
The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell-cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph-ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph-Eph interfaces. One Eph-Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor-receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.
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Receptor EphB2/química , Receptor EphB2/metabolismo , Transducción de Señal , Animales , Células HEK293 , Humanos , Ratones , Dominios Proteicos , Receptor EphB2/genética , Relación Estructura-ActividadRESUMEN
More than 3.5 million people have died globally from COVID-19, yet an effective therapy is not available. It is, therefore, important to understand the signaling pathways that mediate disease progression in order to identify new molecular targets for therapeutic development. Here, we report that the blood serum levels of ephrin-A1 and the sheddase ADAM12 were significantly elevated in COVID-19 patients treated at SUNY Downstate Hospital of Brooklyn, New York. Both ephrin-A1 and ADAM12 are known to be involved in inflammation and regulate endothelial cell permeability, thus providing a gateway to lung injury. The clinical outcome correlated with the ephrin-A1 and ADAM12 serum levels during the first week of hospitalization. In contrast, the serum levels of TNFα were elevated in only a small subset of the patients, and these same patients also had highly elevated levels of the sheddase ADAM17. These data indicate that ephrin-A1-mediated inflammatory signaling may contribute to COVID-19 disease progression more so than TNFα-mediated inflammatory signaling. They also support the notion that, in COVID-19 inflammation, ADAM12 sheds ephrin-A1, while ADAM17 sheds TNFα. Furthermore, the results suggest that elevated serum levels and activity of cytokines, such as TNFα, and other secreted inflammatory molecules, such as ephrin-A1, are not simply due to overexpression, but also to upregulation of sheddases that release them into the blood circulation. Our results identify ephrin-A1, ADAM12, and other molecules in the ephrin-A1 signaling pathway as potential pharmacological targets for treating COVID-19 inflammation.
RESUMEN
Eph receptors are the largest group amongst the receptor tyrosine kinases and are divided into two subgroups, A and B, based on ligand binding specificities and sequence conservation. Through ligand-induced and ligand-independent activities, Ephs play central roles in diverse biological processes, including embryo development, regulation of neuronal signaling, immune responses, vasculogenesis, as well as tumor initiation, progression, and metastasis. The Eph extracellular regions (ECDs) are constituted of multiple domains, and previous structural studies of the A class receptors revealed how they interact with ephrin ligands and simultaneously mediate Eph-Eph clustering necessary for biological activity. Specifically, EphA structures highlighted a model, where clustering of ligand-bound receptors relies on two distinct receptor/receptor interfaces. Interestingly, most unliganded A class receptors also form an additional, third interface, between the ligand binding domain (LBD) and the fibronectin III domain (FN3) of neighboring molecules. Structures of B-class Eph ECDs, on the other hand, have never been reported. To further our understanding of Eph receptor function, we crystallized the EphB6-ECD and determined its three-dimensional structure using X-ray crystallography. EphB6 has important functions in both normal physiology and human malignancies and is especially interesting because this atypical receptor innately lacks kinase activity and our understanding of the mechanism of action is still incomplete. Our structural data reveals the overall EphB6-ECD architecture and shows EphB6-LBD/FN3 interactions similar to those observed for the unliganded A class receptors, suggesting that these unusual interactions are of general importance to the Eph group. We also observe unique structural features, which likely reflect the atypical signaling properties of EphB6, namely the need of co-receptor(s) for this kinase-inactive Eph. These findings provide new valuable information on the structural organization and mechanism of action of the B-class Ephs, and specifically EphB6, which in the future will assist in identifying clinically relevant targets for cancer therapy.
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Receptor EphB6/ultraestructura , Receptores de la Familia Eph/ultraestructura , Línea Celular , Cristalografía por Rayos X/métodos , Efrinas/metabolismo , Fibronectinas/metabolismo , Humanos , Ligandos , Fosforilación , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Receptor EphA1/metabolismo , Receptor EphA1/ultraestructura , Receptor EphB6/metabolismo , Receptores de la Familia Eph/metabolismo , Transducción de SeñalRESUMEN
Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.
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Anticuerpos Antivirales/uso terapéutico , COVID-19/terapia , SARS-CoV-2/inmunología , Adulto , Anciano , Anticuerpos Antivirales/inmunología , Donantes de Sangre , Selección de Donante , Femenino , Humanos , Inmunización Pasiva/métodos , Inmunoglobulina G/sangre , Inmunoglobulina G/uso terapéutico , Inmunoglobulina M/sangre , Inmunoglobulina M/uso terapéutico , Masculino , Persona de Mediana Edad , Plasma/inmunología , Estudios Retrospectivos , Sueroterapia para COVID-19RESUMEN
ADAM proteases are multi domain transmembrane metalloproteases that cleave a range of cell surface proteins and activate signaling pathways implicated in tumor progression, including those mediated by Notch, EFGR, and the Eph receptors. Consequently, they have emerged as key therapeutic targets in the efforts to inhibit tumor initiation and progression. To that end, two main approaches have been taken to develop ADAM antagonists: (i) small molecule inhibitors, and (ii) monoclonal antibodies. In this mini-review we describe the distinct features of ADAM proteases, particularly of ADAM10 and ADAM17, their domain organization, conformational rearrangements, regulation, as well as their emerging importance as therapeutic targets in cancer. Further, we highlight an anti-ADAM10 monoclonal antibody that we have recently developed, which has shown significant promise in inhibiting Notch signaling and deterring growth of solid tumors in pre-clinical settings.
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Proteínas ADAM/química , Proteínas ADAM/metabolismo , Neoplasias/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM10/antagonistas & inhibidores , Proteína ADAM10/química , Proteína ADAM10/metabolismo , Proteína ADAM17/química , Proteína ADAM17/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Dominio Catalítico , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Conformación Proteica , Dominios ProteicosRESUMEN
Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only â¼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.
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Efrina-B1/metabolismo , Efrina-B2/metabolismo , Efrina-B3/metabolismo , Infecciones por Henipavirus/virología , Henipavirus/fisiología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química , Animales , Fusión Celular , Efrina-B1/genética , Efrina-B2/genética , Efrina-B3/genética , Infecciones por Henipavirus/genética , Infecciones por Henipavirus/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , Conformación Proteica , Receptores Virales/genética , Especificidad de la Especie , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
The Eph-ephrin signaling pathway mediates developmental processes and the proper functioning of the adult human body. This distinctive bidirectional signaling pathway includes a canonical downstream signal cascade inside the Eph-bearing cells, as well as a reverse signaling in the ephrin-bearing cells. The signaling is terminated by ADAM metalloproteinase cleavage, internalization, and degradation of the Eph/ephrin complexes. Consequently, the Eph-ephrin-ADAM signaling cascade has emerged as a key target with immense therapeutic potential particularly in the context of cancer. An interesting twist was brought forth by the emergence of ephrins as the entry receptors for the pathological Henipaviruses, which has spurred new studies to target the viral entry. The availability of high-resolution structures of the multi-modular Eph receptors in complexes with ephrins and other binding partners, such as peptides, small molecule inhibitors and antibodies, offers a wealth of information for the structure-guided development of therapeutic intervention. Furthermore, genomic data mining of Eph mutants involved in cancer provides information for targeted drug development. In this review we summarize the distinct avenues for targeting the Eph-ephrin signaling pathway, including its termination by ADAM proteinases. We highlight the latest developments in Eph-related pharmacology in the context of Eph-ephrin-ADAM-based antibodies and small molecules. Finally, the future prospects of genomics- and proteomics-based medicine are discussed.
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Efrinas/efectos de los fármacos , Efrinas/metabolismo , Receptores de la Familia Eph/efectos de los fármacos , Receptores de la Familia Eph/metabolismo , Proteínas ADAM/efectos de los fármacos , Proteínas ADAM/metabolismo , Anticuerpos/química , Anticuerpos/farmacología , Antineoplásicos/farmacología , Sitios de Unión , Desarrollo de Medicamentos , Efrinas/química , Humanos , Modelos Biológicos , Modelos Moleculares , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Receptores de la Familia Eph/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.
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Efrina-A5/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Línea Celular , Efrina-A5/química , Técnicas In Vitro , Ratones , Neuronas Motoras/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-ret/química , Receptor EphA4/metabolismo , Células Sf9 , Proteínas de Pez Cebra/químicaRESUMEN
Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.
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Proteína ADAM10/química , Secretasas de la Proteína Precursora del Amiloide/química , Proteínas de la Membrana/química , Proteolisis , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance.
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Proteína ADAM10/fisiología , Neoplasias Experimentales/patología , Células Madre Neoplásicas/patología , Proteína ADAM10/antagonistas & inhibidores , Proteína ADAM10/química , Proteína ADAM17/fisiología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Receptores Notch/fisiologíaRESUMEN
Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.
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Infecciones por Henipavirus/metabolismo , Virus Nipah/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Virus Nipah/metabolismo , Conformación Proteica , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The amyloid precursor protein (APP) has garnered considerable attention due to its genetic links to Alzheimer's disease. Death receptor 6 (DR6) was recently shown to bind APP via the protein extracellular regions, stimulate axonal pruning, and inhibit synapse formation. Here, we report the crystal structure of the DR6 ectodomain in complex with the E2 domain of APP and show that it supports a model for APP-induced dimerization and activation of cell surface DR6.
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Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Moleculares , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Cristalización , Dimerización , Células HEK293 , Humanos , Ratones , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, and mediate many other cell-cell communication events. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. Recent structural, biochemical and cell-biological studies revealed that these assemblies are generated not only via Eph-ephrin interactions, but also via homotypic interactions between neighboring receptor molecules. In addition, Eph-Eph interactions mediate receptor pre-clustering, which ensures fast and efficient activation once ligands come into contact range. Here we summarize the current knowledge about the homotypic Eph-Eph interactions and discuss how they could modulate the initiation of Eph/ephrin signaling.
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Comunicación Celular , Efrinas/metabolismo , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Movimiento Celular , Humanos , Ligandos , Unión ProteicaRESUMEN
Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy.