RESUMO
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologiaRESUMO
Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.
Assuntos
Membrana Celular/química , Proteínas Hedgehog/agonistas , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Esteróis/farmacologia , Animais , Sítios de Ligação , Técnicas Biossensoriais , Domínio Catalítico/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacologia , Proteínas Hedgehog/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Receptor Patched-1/antagonistas & inibidores , Receptor Patched-1/metabolismo , Conformação Proteica , Estabilidade Proteica , Anticorpos de Cadeia Única/imunologia , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/química , Esteróis/química , Esteróis/metabolismo , Proteínas de Xenopus/químicaRESUMO
Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular/química , DNA Fúngico/química , DNA de Cadeia Simples/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Proteínas Serina-Treonina Quinases/química , Proteína de Replicação A/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação de Sentido Incorreto , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Receptor Smoothened/fisiologia , Animais , Animais Geneticamente Modificados , Domínio Catalítico/genética , Células Cultivadas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Embrião não Mamífero , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/genética , Receptor Smoothened/metabolismo , Peixe-ZebraRESUMO
The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Complexos Multiproteicos/metabolismo , Animais , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Epistasia Genética , Exodesoxirribonucleases , Proteína de Replicação A , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.
Assuntos
Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Heterocromatina/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Humanos , Proteínas Hedgehog/genética , Receptores Acoplados a Proteínas G/metabolismo , Mutação , Cílios/metabolismoRESUMO
The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.
RESUMO
The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.
Assuntos
Dinoprostona , Receptores de Prostaglandina , Humanos , Camundongos , Animais , Fagocitose , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dor/tratamento farmacológico , Anti-Inflamatórios não Esteroides/farmacologiaRESUMO
The Hedgehog signaling pathway coordinates early development and is important in various cancers. Classic approaches to test pathway activation rely on transcriptional readouts or ciliary accumulation of specific pathway components. Although these assays have laid the foundation for studying Hedgehog pathway activation, they integrate the complex molecular actions of the transporter Patched and the seven transmembrane protein Smoothened. Though it is clear that cellular sterols are critical for pathway activity, direct dissection of which sterols drive Smoothened activity is precluded by the complex biosynthetic pathways responsible for cellular sterols. Here we describe a direct method of measuring Smoothened activity in vitro. This assay measures the binding of Smoothened to NbSmo8, a single-domain antibody that is selective for the active-state of Smoothened. Binding of purified Smoothened, reconstituted with specific sterols, to fluorescently labeled NbSmo8 can be rapidly evaluated using a fluorescence-detection size-exclusion chromatography assay. This approach enables a reductionist approach to precisely interrogate the regulatory activities of cellular lipids and sterols during Hedgehog signaling.
Assuntos
Receptor Smoothened/metabolismo , Proteínas Hedgehog , Proteínas de Membrana , Receptores Acoplados a Proteínas G , Transdução de Sinais , EsteróisRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
Assuntos
Domínio Catalítico/fisiologia , Ligação Proteica/fisiologia , Proteínas não Estruturais Virais/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/genética , Tratamento Farmacológico da COVID-19RESUMO
Substantial number of breast cancer (BC) patients undergoing radiation therapy (RT) develop local recurrence over time. During RT therapy, cells can gradually acquire resistance implying adaptive radioresistance. Here we probe the mechanisms underlying this acquired resistance by first establishing radioresistant lines using ZR-75-1 and MCF-7 BC cells through repeated exposure to sub-lethal fractionated dose of 2Gy up to 15 fractions. Radioresistance was found to be associated with increased cancer stem cells (CSCs), and elevated EpCAM expression in the cell population. A retrospective analysis of TCGA dataset indicated positive correlation of high EpCAM expression with poor response to RT. Intriguingly, elevated EpCAM expression in the radioresistant CSCs raise the bigger question of how this biomarker expression contributes during radiation treatment in BC. Thereafter, we establish EpCAM overexpressing ZR-75-1 cells (ZR-75-1EpCAM), which conferred radioresistance, increased stemness through enhanced AKT activation and induced a hybrid epithelial/mesenchymal phenotype with enhanced contractility and invasiveness. In line with these observations, orthotopic implantation of ZR-75-1EpCAM cells exhibited faster growth, lesser sensitivity to radiation therapy and increased lung metastasis than baseline ZR-75-1 cells in mice. In summary, this study shows that similar to radioresistant BC cells, EpCAM overexpressing cells show high degree of plasticity and heterogeneity which ultimately induces radioresistant and metastatic behavior of cancer cells, thus aggravating the disease condition.
RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Afinidade de Anticorpos , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , Testes de Neutralização , Ligação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/química , Glicoproteína da Espícula de Coronavírus/química , Células VeroRESUMO
The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
RESUMO
Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.