RESUMO
The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.
Assuntos
Adipocinas , Leptina , Leptina/genética , Leptina/metabolismo , Leptina/farmacologia , Isoformas de Proteínas/genética , Transdução de SinaisRESUMO
BACKGROUND: Parameters from maximal expiratory flow-volume curves (MEFVC) have been linked to CT-based parameters of COPD. However, the association between MEFVC shape and phenotypes like emphysema, small airways disease (SAD) and bronchial wall thickening (BWT) has not been investigated. RESEARCH QUESTION: We analyzed if the shape of MEFVC can be linked to CT-determined emphysema, SAD and BWT in a large cohort of COPDGene participants. STUDY DESIGN AND METHODS: In the COPDGene cohort, we used principal component analysis (PCA) to extract patterns from MEFVC shape and performed multiple linear regression to assess the association of these patterns with CT parameters over the COPD spectrum, in mild and moderate-severe COPD. RESULTS: Over the entire spectrum, in mild and moderate-severe COPD, principal components of MEFVC were important predictors for the continuous CT parameters. Their contribution to the prediction of emphysema diminished when classical pulmonary function test parameters were added. For SAD, the components remained very strong predictors. The adjusted R2 was higher in moderate-severe COPD, while in mild COPD, the adjusted R2 for all CT outcomes was low; 0.28 for emphysema, 0.21 for SAD and 0.19 for BWT. INTERPRETATION: The shape of the maximal expiratory flow-volume curve as analyzed with PCA is not an appropriate screening tool for early disease phenotypes identified by CT scan. However, it contributes to assessing emphysema and SAD in moderate-severe COPD.
Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Análise de Componente Principal , Fumar , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/genética , Espirometria , Fenótipo , Volume Expiratório ForçadoRESUMO
Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.
Assuntos
Fragmentos de Imunoglobulinas/metabolismo , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular Tumoral , Criança , Endocitose , Células HEK293 , Humanos , Camundongos , Fosforilação , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.
Assuntos
Glicoproteínas , Schistosoma mansoni , Animais , Proteínas de Ligação a DNA , Epitopos/química , Fucose/metabolismo , Glicoproteínas/metabolismo , Humanos , Imunoglobulina M , Mamíferos/metabolismo , Proteínas de Membrana , Camundongos , Polissacarídeos/química , Schistosoma mansoni/química , Schistosoma mansoni/metabolismoAssuntos
Pólipos Nasais , Neutrófilos , Armadilhas Extracelulares/imunologia , Feminino , Galectinas/imunologia , Humanos , Inflamassomos/imunologia , Inflamação , Interleucina-1beta/imunologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Pólipos Nasais/imunologia , Pólipos Nasais/patologia , Neutrófilos/imunologia , Neutrófilos/patologiaRESUMO
Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Asma/terapia , Glicoproteínas/química , Glicoproteínas/farmacologia , Imunidade Inata/efeitos dos fármacos , Lisofosfolipase/química , Lisofosfolipase/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/terapia , Cristalização , Modelos Animais de Doenças , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Células Caliciformes/imunologia , Células Caliciformes/patologia , Humanos , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Lisofosfolipase/administração & dosagem , Lisofosfolipase/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Muco/imunologiaRESUMO
Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.
Assuntos
ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Ciclo do Ácido Cítrico , Evolução Molecular , ATP Citrato (pro-S)-Liase/genética , Biocatálise , Chlorobium/enzimologia , Chlorobium/genética , Cristalografia por Raios X , Humanos , Methanosarcinales/enzimologia , Methanosarcinales/genética , Modelos MolecularesRESUMO
Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucina-2/imunologia , Parapoxvirus/imunologia , Proteínas Virais/imunologia , Cristalografia por Raios X , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-2/química , Interleucina-2/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Parapoxvirus/metabolismo , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/metabolismo , Infecções por Poxviridae/virologia , Ligação Proteica , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Human colony-stimulating factor 1 receptor (hCSF-1R) is unique among the hematopoietic receptors because it is activated by two distinct cytokines, CSF-1 and interleukin-34 (IL-34). Despite ever-growing insights into the central role of hCSF-1R signaling in innate and adaptive immunity, inflammatory diseases, and cancer, the structural basis of the functional dichotomy of hCSF-1R has remained elusive. Here, we report crystal structures of ternary complexes between hCSF-1 and hCSF-1R, including their complete extracellular assembly, and propose a mechanism for the cooperative human CSF-1:CSF-1R complex that relies on the adoption by dimeric hCSF-1 of an active conformational state and homotypic receptor interactions. Furthermore, we trace the cytokine-binding duality of hCSF-1R to a limited set of conserved interactions mediated by functionally equivalent residues on CSF-1 and IL-34 that play into the geometric requirements of hCSF-1R activation, and map the possible mechanistic consequences of somatic mutations in hCSF-1R associated with cancer.
Assuntos
Cristalografia por Raios X , Fator Estimulador de Colônias de Macrófagos/química , Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Sítios de Ligação , Ativação Enzimática , Humanos , Modelos Moleculares , Fosforilação , Espalhamento a Baixo Ângulo , Transdução de Sinais , Difração de Raios XRESUMO
Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.
Assuntos
Interleucina-23/antagonistas & inibidores , Peptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/uso terapêutico , Psoríase/prevenção & controleRESUMO
Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-I-leg of GSSG is accommodated tightly via extensive interactions contributed by the N- and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/metabolismo , Bactérias Gram-Positivas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Streptococcus mutans/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sítios de Ligação , Transporte Biológico , Cristalografia , Cistina/metabolismo , Glutationa/análogos & derivados , Glutationa/química , Dissulfeto de Glutationa/metabolismo , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Streptococcus mutans/química , Streptococcus mutans/genética , Streptococcus mutans/crescimento & desenvolvimentoRESUMO
Intracellular signalling cascades initiated by class III receptor tyrosine kinases (RTK-IIIs) and their cytokine ligands contribute to haematopoiesis and mesenchymal tissue development. They are also implicated in a wide range of inflammatory disorders and cancers. Recent snapshots of RTK-III ectodomains in complex with cognate cytokines have revealed timely insights into the structural determinants of RTK-III activation, evolution and pathology. Importantly, candidate 'driver' and 'passenger' mutations that have been identified in RTK-IIIs can now be collectively mapped for the first time to structural scaffolds of the corresponding RTK-III ectodomains. Such insights will generate a renewed interest in dissecting the mechanistic effects of such mutations and their therapeutic relevance.
Assuntos
Citocinas/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Transformação Celular Neoplásica , Hematopoese/fisiologia , Humanos , Ligantes , Mutação , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor-binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Fator Estimulador de Colônias de Macrófagos/metabolismo , Monócitos/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Herpesvirus Humano 4/metabolismo , Humanos , Fator Estimulador de Colônias de Macrófagos/química , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Monócitos/citologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transdução de Sinais , Proteínas Virais/químicaRESUMO
The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex. Studies by small-angle X-ray scattering of unliganded hCSF-1R point to large domain rearrangements upon CSF-1 binding, and provide structural evidence for the relevance of receptor predimerization at the cell surface. Comparative structural and binding studies aiming to dissect the assembly principles of human and mouse CSF-1R complexes, including a quantification of the CSF-1/CSF-1R species cross-reactivity, show that bivalent cytokine binding to receptor coupled to ensuing receptor-receptor interactions are common denominators in extracellular complex formation.
Assuntos
Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Animais , Sítios de Ligação , Sistema Hematopoético , Humanos , Ligantes , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Microscopia Eletrônica , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , TermodinâmicaRESUMO
The class III receptor tyrosine kinase (RTKIII) Fms-like tyrosine kinase receptor 3 (Flt3) and its cytokine ligand (FL) play central roles in hematopoiesis and the immune system, by establishing signaling cascades crucial for the development and homeostasis of hematopoietic progenitors and antigen-presenting dendritic cells. However, Flt3 is also one of the most frequently mutated receptors in hematologic malignancies and is currently a major prognostic factor and clinical target for acute myeloid leukemia. Here, we report the structural basis for the Flt3 ligand-receptor complex and unveil an unanticipated extracellular assembly unlike any other RTKIII/V complex characterized to date. FL induces dimerization of Flt3 via a remarkably compact binding epitope localized at the tip of extracellular domain 3 of Flt3, and it invokes a ternary complex devoid of homotypic receptor interactions. Comparisons of Flt3 with homologous receptors and available mutagenesis data for FL have allowed us to rationalize the unique features of the Flt3 extracellular assembly. Furthermore, thermodynamic dissection of complex formation points to a pronounced enthalpically driven binding event coupled to an entropic penalty. Together, our data suggest that the high-affinity Flt3:FL complex is driven in part by a single preformed binding epitope on FL reminiscent of a "lock-and-key" binding mode, thereby setting the stage for antagonist design.
Assuntos
Citocinas/química , Citocinas/metabolismo , Hematopoese/fisiologia , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms , Sequência de Aminoácidos , Cristalografia por Raios X , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Relação Estrutura-Atividade , Termodinâmica , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of antigen-presenting dendritic cells and CD34(+) natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.