RESUMO
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/terapia , Mastócitos/enzimologia , Mastócitos/imunologia , Triptases/antagonistas & inibidores , Triptases/imunologia , Adolescente , Regulação Alostérica/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , CoelhosRESUMO
BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.
Assuntos
Asma , Estudo de Associação Genômica Ampla , Anticorpos Neutralizantes/genética , Asma/genética , Quimiocinas/genética , Citocinas/metabolismo , Humanos , Inflamação/genética , Interleucina-13/genética , Interleucina-4/genética , Calicreínas/genética , Calicreínas/metabolismoRESUMO
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic ß-tryptase inhibitors, but its unique activation mechanism is less well-explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N terminus into its "activation pocket," indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric ß-tryptase mutant (I99C*/Y75A/Y37bA, where C* is cysteinylated Cys-99) cannot form a dimer or tetramer, yet it is active but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each ß-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.
Assuntos
Heparina/metabolismo , Peptídeo Hidrolases/metabolismo , Regiões Promotoras Genéticas , Multimerização Proteica , Triptases/genética , Triptases/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Subunidades Proteicas , Triptases/químicaRESUMO
Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like ß-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Peptídeos/farmacologia , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Humanos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/síntese química , Ligação Proteica , Proteína C/química , Proteína C/genética , Proteína C/metabolismo , Engenharia de Proteínas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.
Assuntos
Dermatite Atópica , Síndrome de Netherton , Dermatopatias , Camundongos , Humanos , Animais , Síndrome de Netherton/genética , Síndrome de Netherton/metabolismo , Síndrome de Netherton/patologia , Dermatite Atópica/patologia , Inibidor de Serinopeptidase do Tipo Kazal 5/metabolismo , Epiderme/patologia , Dermatopatias/metabolismo , Anticorpos/metabolismo , Calicreínas/metabolismoRESUMO
Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/ß-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like ß-chain (HGF ß), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the ß-chain stimulate Met phosphorylation by pro-HGF to levels that are â¼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF ß binding to Met, resulting in a K(D)(app) of 1.6 µm, only 8-fold weaker than the Met/HGF ß-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like ß-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Oligopeptídeos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Substituição de Aminoácidos , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Fator de Crescimento de Hepatócito/genética , Humanos , Mutação de Sentido Incorreto , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Precursores de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento/genética , Serina Proteases/genética , Serina Proteases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10-20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn(2+) cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.
Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Cálcio/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Epitopos , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Ligação Proteica , Engenharia de ProteínasRESUMO
Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked alpha/beta-heterodimer having a trypsin serine protease-like beta-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn(2+) metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met beta-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation.
Assuntos
Proteínas Hedgehog/química , Proteínas Hedgehog/fisiologia , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/fisiologia , Neoplasias/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Animais , Proteínas de Transporte/metabolismo , Domínio Catalítico , Desenho de Fármacos , Proteínas Hedgehog/antagonistas & inibidores , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Humanos , Ligantes , Glicoproteínas de Membrana/metabolismo , Metaloproteases/química , Neoplasias/tratamento farmacológico , Receptores Patched , Receptor Patched-1 , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Serina Proteases/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.
Assuntos
Imunoensaio/métodos , Testes Imunológicos/métodos , Mastócitos/patologia , Triptases/metabolismo , HumanosRESUMO
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.
Assuntos
Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Triptases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Heparina/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Modelos Moleculares , Proteínas Mutantes/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Triptases/químicaRESUMO
The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)(27-268) at 2.65 A resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp(159) in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu).
Assuntos
Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteína GLI1 em Dedos de ZincoRESUMO
Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.
Assuntos
Dissulfetos/metabolismo , Fator VIIa/metabolismo , Animais , Células CHO , Cromatografia de Afinidade , Cricetinae , Dissulfetos/química , Fator VIIa/química , Cinética , Mutagênese , Plasmídeos , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn's disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis. METHODS: RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles. RESULTS: In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum. CONCLUSIONS: By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.
Assuntos
Alelos , Predisposição Genética para Doença , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Polimorfismo Genético , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/sangue , Humanos , Doenças Inflamatórias Intestinais/patologia , Intestinos/patologia , Camundongos , Modelos Moleculares , Conformação Proteica , Proteólise , Proteínas Proto-Oncogênicas/sangue , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity. We report here a method that can be easily and generally applied to sorting of phage display libraries with multispan protein targets solubilized in detergent. A key feature of this approach is the production of biotinylated multispan proteins in virions of a baculovirus vector that allows library panning without prior purification of the target protein. We obtained Fab fragments from a naïve synthetic antibody phage library that, when engineered into full-length immunoglobulin (Ig)G, specifically bind cells expressing claudin-1, a protein with four transmembrane regions that is used as an entry co-receptor by the hepatitis C virus (HCV). Affinity-matured variants of one of these antibodies efficiently inhibited HCV infection. The use of baculovirus particles as a source of mammalian multispan protein facilitates the application of phage display to this difficult class of proteins.
Assuntos
Baculoviridae/genética , Fragmentos Fab das Imunoglobulinas/biossíntese , Imunoglobulina G/biossíntese , Proteínas de Membrana/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Linhagem Celular Tumoral , Claudina-1 , Citometria de Fluxo , Células HEK293 , Hepacivirus , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Ligação Proteica , Alinhamento de Sequência , Estreptavidina , Vírion/química , Vírion/metabolismoRESUMO
The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.
Assuntos
Arthrobacter/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Cristalografia por Raios X , Modelos Moleculares , Nicotina/análogos & derivados , Nicotina/metabolismo , Ácidos Fosfatídicos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/químicaRESUMO
Hedgehog (Hh) signaling is crucial for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hedgehog-interacting protein (Hhip) is a surface receptor antagonist that is equipotent against all three mammalian Hh homologs. The crystal structures of human HHIP alone and bound to Sonic hedgehog (SHH) now reveal that HHIP is comprised of two EGF domains and a six-bladed beta-propeller domain. In the complex structure, a critical loop from HHIP binds the pseudo active site groove of SHH and directly coordinates its Zn2+ cation. Notably, sequence comparisons of this SHH binding loop with the Hh receptor Patched (Ptc1) ectodomains and HHIP- and PTC1-peptide binding studies suggest a 'patch for Patched' at the Shh pseudo active site; thus, we propose a role for Hhip as a structural decoy receptor for vertebrate Hh.
Assuntos
Proteínas de Transporte/química , Proteínas Hedgehog/química , Glicoproteínas de Membrana/química , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Domínio Catalítico , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Receptores Patched , Conformação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked alpha/beta-heterodimer, where the beta-chain of HGF (HGF beta) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the beta-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF beta crystal structure shows that V495 inserts into the "activation pocket" near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF beta domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF alpha-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Serina Endopeptidases/metabolismo , Sítio Alostérico , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/química , Transdução de SinaisRESUMO
Limitations of current anticoagulant therapies have led us to develop two distinct classes of exosite peptide inhibitors for the initiator of the clotting process, the tissue factor-factor VIIa (TF.FVIIa) complex (Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531). Although both peptide classes are potent and selective inhibitors of TF.FVIIa, neither showed 100% inhibition at saturating concentrations. Crystal structures of these peptides in complex with the FVII/FVIIa protease domain revealed their distinct binding sites and close proximity to the active site. The favorable orientation of the 15-mer A-site peptide A-183 (EEWEVLCWTWETCER) suggested that a C-terminal extension into the FVIIa active site could yield a chimeric inhibitor that was not only potent and selective but complete as well. A novel two-step "protease switch" approach using substrate phage display was developed by first binding all phage containing A-183 and C-terminal extension libraries to immobilized and inactive FVIIa. Upon altering pH and adding TF to switch on FVIIa enzymatic activity, only those phage released by proteolytic cleavage within the extension were propagated. This process selected for both preferred sequence and length in the extension, leading to a 27-mer peptide A-183X (EEWEVLCWTWETCERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of approximately 99% with an IC50 of 230 pm versus A-183 which maximally inhibited to 74% with an IC50 of 1.5 nm. A-183X also had a maximal prolongation of the prothrombin time of 7.6- versus 1.9-fold for A-183, making it a more effective anticoagulant.