RESUMEN

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

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/metabolismo

RESUMEN

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/metabolismo

RESUMEN

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/química

RESUMEN

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.

Asunto(s)

Inmunoensayo/métodos , Pruebas Inmunológicas/métodos , Mastocitos/patología , Triptasas/metabolismo , Humanos

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 , Conejos

RESUMEN

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/metabolismo

RESUMEN

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/química

Asunto(s)

Ingeniería de Proteínas , Serina Endopeptidasas/uso terapéutico , Factor de von Willebrand/uso terapéutico , Minería de Datos , Humanos , Serina Endopeptidasas/genética , Factor de von Willebrand/genética

RESUMEN

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/metabolismo

RESUMEN

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/genética

RESUMEN

ß-Secretase (BACE1) is a membrane-anchored pepsin-like aspartic protease and is the rate-limiting enzyme in the ß-amyloidogenic pathway. Thus, inhibitors of BACE1 activity have therapeutic potential for Alzheimer's disease. While much effort has focused on small molecule active site inhibitors, recent exploration of BACE1 inhibition by peptides and antibodies has revealed exosites that can regulate enzymatic activity. This type of allosteric regulation by proteinaceous factors, while frequently found in serine and cysteine proteases, is rarely seen in aspartic proteases. A crystal structure of the anti-BACE1/enzyme complex shows altered structural features and dynamic characteristics near the substrate-binding cleft. This binding mode, along with the enzymatic inhibition pattern, suggests that anti-BACE1 functions through an allosteric inhibition mechanism.

Asunto(s)

Anticuerpos/inmunología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/inmunología , Regulación Alostérica , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Diseño de Fármacos , Humanos , Inhibidores de Proteasas/farmacología

RESUMEN

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 Aminoácido

RESUMEN

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.

Asunto(s)

Alelos , Predisposición Genética a la Enfermedad , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Polimorfismo Genético , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/sangre , Humanos , Enfermedades Inflamatorias del Intestino/patología , Intestinos/patología , Ratones , Modelos Moleculares , Conformación Proteica , Proteolisis , Proteínas Proto-Oncogénicas/sangre , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal

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 , Viscosidad

RESUMEN

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/ß heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg(483)-Val(484) with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON-Fc fusion protein, whereas hepsin-cleaved MSP bound with a K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP ß-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.

Asunto(s)

Neoplasias Ováricas/metabolismo , Neoplasias de la Próstata/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Precursores de Proteínas/genética , Proteolisis , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal

RESUMEN

Reducing production of amyloid-ß (Aß) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer's disease. However, small-molecule inhibitors of the ß-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aß production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aß peptide concentrations. Anti-BACE1 also reduces central nervous system Aß concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer's disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/deficiencia , Ácido Aspártico Endopeptidasas/metabolismo , Bioensayo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cristalografía por Rayos X , Endocitosis/efectos de los fármacos , Humanos , Macaca fascicularis , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos , Resultado del Tratamiento

RESUMEN

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 ∼25% 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 fármacos

RESUMEN

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 Señal