RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/terapia , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/antagonistas & inhibidores , Triptasas/inmunología , Adolescente , Regulación Alostérica/inmunología , Animales , Línea Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ConejosRESUMEN
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.
Asunto(s)
Asma , Estudio de Asociación del Genoma Completo , Anticuerpos Neutralizantes/genética , Asma/genética , Quimiocinas/genética , Citocinas/metabolismo , Humanos , Inflamación/genética , Interleucina-13/genética , Interleucina-4/genética , Calicreínas/genética , Calicreínas/metabolismoRESUMEN
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.
Asunto(s)
Heparina/metabolismo , Péptido Hidrolasas/metabolismo , Regiones Promotoras Genéticas , Multimerización de Proteína , Triptasas/genética , Triptasas/metabolismo , Regulación Alostérica , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Subunidades de Proteína , Triptasas/químicaRESUMEN
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.
Asunto(s)
Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Receptores Patched , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Péptidos/farmacología , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células CHO , Dominio Catalítico , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetulus , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/genética , Humanos , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Proteína C/química , Proteína C/genética , Proteína C/metabolismo , Ingeniería de Proteínas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
INTRODUCTION: The availability of cystic fibrosis transmembrane conductance regulator (CFTR) modulators opens the possibility of discontinuing some chronic pulmonary therapies to decrease cystic fibrosis (CF) treatment burden. However, CFTR modulators may not adequately address neutrophilic inflammation, which contributes to a self-perpetual cycle of viscous CF sputum, airway obstruction, inflammation, and lung function decline. AREAS COVERED: This review discusses the emerging role of neutrophil extracellular traps in CF and its role in CF sputum viscosity, airway obstruction, and inflammation, based on a literature search of PubMed (1990-present). We summarize clinical trials and real-world studies that support the efficacy of dornase alfa (Pulmozyme) in improving lung function and reducing pulmonary exacerbation in people with CF (PwCF), and we discuss the potential role of dornase alfa in reducing airway inflammation. We also examine the findings of short-term trials evaluating the discontinuation of mucoactive therapy in PwCF receiving CFTR modulators. EXPERT OPINION: Long-term studies are needed to assess the impact of discontinuing mucoactive therapy in PwCF who are clinically stable while receiving CFTR modulatory therapy. Treatment decisions should take into account the severity of underlying lung disease. People with advanced CF will likely require ongoing mucoactive therapy.
RESUMEN
Recombinant human DNase I (Pulmozyme, dornase alfa) is used for the treatment of cystic fibrosis where it improves lung function and reduces the number of exacerbations. The physiological mechanism of action is thought to involve the reduction of the viscoelasticity of cystic fibrosis sputum by hydrolyzing high concentrations of DNA into low-molecular mass fragments. Here we describe the 1.95 Å resolution crystal structure of recombinant human DNase I (rhDNase I) in complex with magnesium and phosphate ions, both bound in the active site. Complementary mutagenesis data of rhDNase I coupled to a comprehensive structural analysis of the DNase I-like superfamily argue for the key catalytic role of Asn7, which is invariant among mammalian DNase I enzymes and members of this superfamily, through stabilization of the magnesium ion coordination sphere. Overall, our combined structural and mutagenesis data suggest the occurrence of a magnesium-assisted pentavalent phosphate transition state in human DNase I during catalysis, where Asp168 may play a key role as a general catalytic base.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Magnesio/metabolismo , Fosfatos/metabolismo , Asparagina/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Desoxirribonucleasa I/genética , Humanos , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ViscosidadRESUMEN
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.
Asunto(s)
Dermatitis Atópica , Síndrome de Netherton , Enfermedades de la Piel , Ratones , Humanos , Animales , Síndrome de Netherton/genética , Síndrome de Netherton/metabolismo , Síndrome de Netherton/patología , Dermatitis Atópica/patología , Inhibidor de Serinpeptidasas Tipo Kazal-5/metabolismo , Epidermis/patología , Enfermedades de la Piel/metabolismo , Anticuerpos/metabolismo , Calicreínas/metabolismoRESUMEN
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.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Oligopéptidos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Sustitución de Aminoácidos , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cricetinae , Cricetulus , Factor de Crecimiento de Hepatocito/genética , Humanos , Mutación Missense , Oligopéptidos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Precursores de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Receptores de Factores de Crecimiento/genética , Serina Proteasas/genética , Serina Proteasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Calcio/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Epítopos , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Unión Proteica , Ingeniería de ProteínasRESUMEN
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.
Asunto(s)
Proteínas Hedgehog/química , Proteínas Hedgehog/fisiología , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/fisiología , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Animales , Proteínas Portadoras/metabolismo , Dominio Catalítico , Diseño de Fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Humanos , Ligandos , Glicoproteínas de Membrana/metabolismo , Metaloproteasas/química , Neoplasias/tratamiento farmacológico , Receptores Patched , Receptor Patched-1 , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Serina Proteasas/química , Transducción de Señal/efectos de los fármacosRESUMEN
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.
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Inmunoensayo/métodos , Pruebas Inmunológicas/métodos , Mastocitos/patología , Triptasas/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/metabolismo , Triptasas/metabolismo , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Heparina/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Modelos Moleculares , Proteínas Mutantes/química , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Triptasas/químicaRESUMEN
Both α-tryptase and ß-tryptase are preferentially expressed by human mast cells, but the purpose of α-tryptase is enigmatic, because its tetramers lack protease activity, whereas ß-tryptase tetramers are active proteases. The monogenic disorder called hereditary α-tryptasemia, due to increased α-tryptase gene copies and protein expression, presents with clinical features such as vibratory urticaria and dysautonomia. We show that heterotetramers composed of 2α- and 2ß-tryptase protomers (α/ß-tryptase) form naturally in individuals who express α-tryptase. α/ß-Tryptase, but not homotetramer, activates protease-activated receptor-2 (PAR2), which is expressed on cell types such as smooth muscle, neurons, and endothelium. Also, only α/ß-tryptase makes mast cells susceptible to vibration-triggered degranulation by cleaving the α subunit of the EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) mechanosensory receptor. Allosteric effects of α-tryptase protomers on neighboring ß-tryptase protomers likely result in the novel substrate repertoire of α/ß-tryptase tetramers that in turn cause some of the clinical features of hereditary α-tryptasemia and of other disorders involving mast cells.
Asunto(s)
Degranulación de la Célula , Enfermedades Genéticas Congénitas , Mastocitos/enzimología , Multimerización de Proteína , Triptasas , Vibración/efectos adversos , Adulto , Regulación Alostérica/genética , Femenino , Enfermedades Genéticas Congénitas/enzimología , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Masculino , Mastocitos/patología , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Triptasas/genética , Triptasas/metabolismoRESUMEN
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).
Asunto(s)
Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Análisis Mutacional de ADN , Regulación de la Expresión Génica/fisiología , Genes Reporteros , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteína con Dedos de Zinc GLI1RESUMEN
While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523-533, 2017.
Asunto(s)
Actinas/química , Clonación Molecular/métodos , Desoxirribonucleasa I/química , Desoxirribonucleasa I/metabolismo , Ingeniería de Proteínas/métodos , Sales (Química)/química , Animales , Células CHO , Proliferación Celular/fisiología , Cricetulus , Desoxirribonucleasa I/genética , Activación Enzimática , Estabilidad de Enzimas , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Glicoproteínas de Membrana/metabolismo , Conformación Proteica , Serina Endopeptidasas , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Calicreína Plasmática/antagonistas & inhibidores , Calicreína Plasmática/química , Calicreína Plasmática/metabolismo , Unión Proteica , Proteínas Inhibidoras de Proteinasas Secretoras , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Especificidad por Sustrato , Tripsina/química , Tripsina/metabolismoRESUMEN
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.