RESUMO
Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.
Assuntos
Fator de Crescimento de Hepatócito , Kringles , Animais , Dimerização , Fator de Crescimento de Hepatócito/metabolismo , Fígado/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-met/agonistas , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Nonalcoholic steatohepatitis (NASH), an advanced stage of nonalcoholic fatty liver disease (NAFLD), is a rapidly growing and global health problem compounded by the current absence of specific treatments. A major limiting factor in the development of new NASH therapies is the absence of models that capture the unique cellular structure of the liver microenvironment and recapitulate the complexities of NAFLD progression to NASH. Organ-on-a-chip platforms have emerged as a powerful approach to dynamically model diseases and test drugs. Herein, we describe a NASH-on-a-chip platform. Four main types of human primary liver cells (hepatocytes [HCs], Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells [HSCs]) were cocultured under microfluidic dynamics. Our chip-based model successfully recapitulated a functional liver cellular microenvironment with stable albumin and urea secretion for at least 2 weeks. Exposing liver chips to a lipotoxic environment led to gradual development of NASH phenotypic characteristics, including intracellular lipid accumulation, hepatocellular ballooning, HSC activation, and elevation of inflammatory and profibrotic markers. Further, exposure of the chip to elafibranor, a drug under study for the therapy of NASH, inhibited the development of NASH-specific hallmarks, causing an ~8-fold decrease in intracellular lipids, a 3-fold reduction in number of ballooned HCs, a significant reduction in HSC activation, and a significant decrease in the levels of inflammatory and profibrotic markers compared with controls. Conclusion: We have successfully developed a microfluidic NASH-on-a-chip platform that recapitulates the main NASH histologic endpoints in a single chip and that can emerge as a powerful noninvasive, human-relevant, in vitro platform to study disease pathogenesis and develop novel anti-NASH drugs.
Assuntos
Técnicas de Cocultura , Dispositivos Lab-On-A-Chip , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Chalconas/farmacologia , Células Endoteliais/citologia , Células Estreladas do Fígado/citologia , Hepatócitos/citologia , Humanos , Inflamação , Células de Kupffer/citologia , Fígado , Propionatos/farmacologiaRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Fator de Crescimento de Hepatócito/agonistas , Sistema Imunitário , Neurônios/citologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/imunologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comportamento Animal , Sobrevivência Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Progressão da Doença , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gliose/metabolismo , Humanos , Interleucina-4/metabolismo , Kringles , Ligantes , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Neurônios Motores/metabolismo , Neurônios/metabolismo , Células de Schwann/metabolismo , Medula Espinal/metabolismo , Linfócitos T/citologiaRESUMO
MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.
Assuntos
Repetição de Anquirina , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo. In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.
RESUMO
The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.
Assuntos
Anticorpos Bloqueadores/isolamento & purificação , Anticorpos Bloqueadores/metabolismo , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/química , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Xenoenxertos , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos Nus , Transplante de Neoplasias , Ligação Proteica , Conformação Proteica , Proto-Oncogene Mas , Resultado do TratamentoRESUMO
In many cancers, aberrant activation of the Met receptor tyrosine kinase leads to dissociation of cells from the primary tumor, causing metastasis. Accordingly, Met is a high-profile target for the development of cancer therapies, and progress has been made through development of small molecule kinase inhibitors and antibodies. However, both approaches pose significant challenges with respect to either target specificity (kinase inhibitors) or the cost involved in treating large patient cohorts (antibodies). Here, we use a fragment-based approach in order to target the protein-protein interaction (PPI) between the α-chain of hepatocyte growth factor/scatter factor (HGF/SF; the NK1 fragment) and its high-affinity binding site located on the Met Sema domain. Surface plasmon resonance was used for initial fragment library screening and hits were developed into larger compounds using substructure (similarity) searches. We identified compounds able to interfere with NK1 binding to Met, disrupt Met signaling, and inhibit tumorsphere generation and cell migration. Using molecular docking, we concluded that some of these compounds inhibit the PPI directly, whereas others act indirectly. Our results indicate that chemical fragments can efficiently target the HGF/SF-Met interface and may be used as building blocks for generating biologically active lead compounds. This strategy may have broad application for the development of a new class of Met inhibitors, namely receptor antagonists, and in general for the development of small molecule PPI inhibitors of key therapeutic targets when structural information is not available.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Fosforilação , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The multi-domain protein InlB (internalin B) from Listeria monocytogenes is an agonist of the human receptor tyrosine kinase MET. Only the internalin domain directly interacts with MET. The internalin domain consists of seven central leucine-rich repeats (LRRs) flanked by an N-terminal helical cap domain and a C-terminal immunoglobulin-like structure. A potential function of the N-terminal cap in receptor binding could so far not be demonstrated by deleting the cap, since the cap is also implicated in nucleating folding of the LRR domain. RESULTS: We generated an InlB variant (YopM-InlB) in which the InlB cap domain was replaced by the unrelated N-terminal capping structure of the LRR protein YopM from Yersinia enterocolitica. The crystal structure of the engineered protein shows that it folds properly. Because the first LRR is structurally closely linked to the cap domain, we exchanged LRR1 along with the cap domain. This resulted in unexpected structural changes extending to LRR2 and LRR3, which are deeply involved in MET binding. As a consequence, the binding of YopM-InlB to MET was substantially weaker than that of wild type InlB. The engineered protein was about one order of magnitude less active in colony scatter assays than wild type InlB. CONCLUSIONS: We obtained a well-behaved InlB variant with an altered N-terminal capping structure through protein design. The reduced affinity for MET precludes a straightforward interpretation of the results from cell-based assays. Still, the engineered hybrid protein induced cell scatter, suggesting that the cap is required for folding and stability of InlB but is not essential for interactions that assemble the signalling-active receptor complex. The cap swap approach described here is clearly applicable to other L. monocytogenes internalins and other LRR proteins such as YopM and may yield useful structure/function correlates within this protein family.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Listeria monocytogenes/metabolismo , Proteínas de Membrana/química , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-met/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The physiological relevance of contacts in crystal lattices often remains elusive. This was also the case for the complex between the invasion protein internalin B (InlB) from Listeria monocytogenes and its host cell receptor, the human receptor tyrosine kinase (RTK) MET. InlB is a MET agonist and induces bacterial host cell invasion. Activation of RTKs generally involves ligand-induced dimerization of the receptor ectodomain. The two currently available crystal structures of the InlB:MET complex show the same arrangement of InlB and MET in a 1:1 complex, but different dimeric 2:2 assemblies. Only one of these 2:2 assemblies is predicted to be stable by a computational procedure. This assembly is mainly stabilized by a contact between the Cap domain of InlB from one and the Sema domain of MET from another 1:1 complex. Here, we probe the physiological relevance of this interaction. We generated variants of the leucine-rich repeat (LRR) protein InlB by inserting an additional repeat between the first and the second LRR. This should allow formation of the 1:1 complex but disrupt the potential 2:2 complex involving the Cap-Sema contact due to steric distortions. A crystal structure of one of the engineered proteins showed that it folded properly. Binding affinity to MET was comparable to that of wild-type InlB. The InlB variant induced MET phosphorylation and cell scatter like wild-type InlB. These results suggest that the Cap-Sema interaction is not physiologically relevant and support the previously proposed assembly, in which a 2:2 InlB:MET complex is built around a ligand dimer.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Proteínas Proto-Oncogênicas c-met/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia , Humanos , Leucina/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-met/metabolismo , Alinhamento de SequênciaRESUMO
The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519-561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed ß-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Mapeamento de Epitopos , Epitopos , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-met/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Uncontrolled cell survival, growth, angiogenesis and metastasis are essential hallmarks of cancer. Genetic and biochemical data have demonstrated that the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, have a causal role in all of these processes, thus providing a strong rationale for targeting these molecules in cancer. Parallel progress in understanding the structure and function of HGF/SF, MET and associated signalling components has led to the successful development of blocking antibodies and a large number of small-molecule MET kinase inhibitors. In this Review, we discuss these advances, as well as results from recent clinical studies that demonstrate that inhibiting MET signalling in several types of solid human tumours has major therapeutic value.
Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-met/genética , Transição Epitelial-Mesenquimal , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de SinaisRESUMO
BACKGROUND & AIMS: Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). METHODS: Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. RESULTS: Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. CONCLUSIONS: 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Engenharia de Proteínas , Animais , Apoptose , Sítios de Ligação , Tetracloreto de Carbono , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Replicação do DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteína Ligante Fas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Hepatectomia , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/cirurgia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Conformação Proteica , Estabilidade Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Fatores de Tempo , UltracentrifugaçãoRESUMO
Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.
Assuntos
Fator de Crescimento de Hepatócito/uso terapêutico , Nanotecnologia , Neovascularização Patológica/tratamento farmacológico , Processamento Alternativo , Sequência de Aminoácidos , Animais , Células Cultivadas , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Nanopartículas/ultraestrutura , Neovascularização Fisiológica/efeitos dos fármacos , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Peixe-ZebraRESUMO
Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF's activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a "closed" monomeric conformation through interdomain interactions between the N- domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptores de Fatores de Crescimento/agonistas , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cristalografia por Raios X , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Humanos , Kringles , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Citometria de Fluxo/métodos , Fator de Crescimento de Hepatócito/química , Proteínas Proto-Oncogênicas/química , Proteínas Recombinantes de Fusão/química , Animais , Células CHO , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Proteínas Ligadas por GPI , Glicosilação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Ativação Enzimática , Humanos , Ligantes , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas c-met , Receptores de Fatores de Crescimento/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade EstáticaRESUMO
The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin fixed and paraffin embedded (FFPE) for histopathologic evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody (mAb), designated MET4, from a panel of MET-avid mAbs, based on its specific staining pattern in FFPE preparations. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability of 6.25% in intra-assay and interassay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histologic subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-MET on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 mAb accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.
Assuntos
Anticorpos Monoclonais , Biomarcadores Tumorais/análise , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/análise , Biomarcadores Tumorais/normas , Feminino , Formaldeído , Glioma , Humanos , Imuno-Histoquímica , Masculino , Neoplasias/química , Neoplasias/diagnóstico , Neoplasias Ovarianas , Inclusão em Parafina , Fixação de TecidosRESUMO
The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallizes as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.
Assuntos
Fator de Crescimento de Hepatócito/química , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Processamento Alternativo , Animais , Linhagem Celular , Movimento Celular , Clonagem Molecular , Replicação do DNA , Dimerização , Cães , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Camundongos , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/química , Listeria monocytogenes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Listeria monocytogenes/genética , Proteínas de Membrana/genética , Modelos Moleculares , Nêutrons , Ligação Proteica , Estrutura Quaternária de Proteína , Proto-Oncogene Mas , Proteínas Repressoras/genética , Espalhamento a Baixo Ângulo , Soluções , Raios XRESUMO
Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.