RESUMEN
Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/agonistas , Receptores de Citocinas/agonistas , Células A549 , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Movimiento Celular/efectos de los fármacos , Dimerización , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Microscopía Fluorescente , Unión Proteica , Proteínas Proto-Oncogénicas c-met/agonistas , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Factor de Crecimiento de Hepatocito/agonistas , Sistema Inmunológico , Neuronas/citología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/inmunología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Conducta Animal , Supervivencia Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perros , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Gliosis/metabolismo , Humanos , Interleucina-4/metabolismo , Kringles , Ligandos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Neuronas Motoras/metabolismo , Neuronas/metabolismo , Células de Schwann/metabolismo , Médula Espinal/metabolismo , Linfocitos T/citologíaRESUMEN
It is hard to bridge the gap between mathematical formulations and biological implementations of Turing patterns, yet this is necessary for both understanding and engineering these networks with synthetic biology approaches. Here, we model a reaction-diffusion system with two morphogens in a monostable regime, inspired by components that we recently described in a synthetic biology study in mammalian cells.1 The model employs a single promoter to express both the activator and inhibitor genes and produces Turing patterns over large regions of parameter space, using biologically interpretable Hill function reactions. We applied a stability analysis and identified rules for choosing biologically tunable parameter relationships to increase the likelihood of successful patterning. We show how to control Turing pattern sizes and time evolution by manipulating the values for production and degradation relationships. More importantly, our analysis predicts that steep dose-response functions arising from cooperativity are mandatory for Turing patterns. Greater steepness increases parameter space and even reduces the requirement for differential diffusion between activator and inhibitor. These results demonstrate some of the limitations of linear scenarios for reaction-diffusion systems and will help to guide projects to engineer synthetic Turing patterns.
Asunto(s)
Modelos Teóricos , Biología Sintética , Animales , Perros , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/metabolismo , Células de Riñón Canino Madin Darby , Unión Proteica , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Taquicininas/antagonistas & inhibidores , Receptores de Taquicininas/metabolismoRESUMEN
Hepatocyte growth factor (HGF) plays a plethora of roles in the progression of many invasive and metastatic cancers. The interaction between tumour cells and their surrounding stromal environment remains a crucial factor governing tumour invasion and metastasis. HGF is primarily synthesised by stromal fibroblasts as an inactive precursor known as pro-HGF. A number of proteases have demonstrated the ability to convert pro-HGF into the biologically active form of HGF, although the two main factors responsible are HGF activator (HGFA) and matriptase. The HGF activation inhibitors (HAI-1 and HAI-2) are two novel Kunitz-type serine protease inhibitors that regulate HGFA and matriptase activity to govern the influence of HGF within the body. Deregulation of HAI expression can lead to shift in the HGF activation/inhibition balance ratio in favour of enhanced HGF production. Therefore, these HGF activation inhibitors may have a direct bearing on cancer invasion and metastasis. This review examines the accumulating evidence on the emerging role and therapeutic potential of HAI-1 and HAI-2 in cancer.
Asunto(s)
Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Serina Proteinasa/farmacología , Línea Celular Tumoral , Factor de Crecimiento de Hepatocito/agonistas , Humanos , Neoplasias/metabolismo , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
Hepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.
Asunto(s)
Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Neuroquinina-1/metabolismo , Animales , Línea Celular , Dimerización , Disulfuros/química , Perros , Escherichia coli/genética , Heparina/farmacología , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Histidina/química , Humanos , Concentración 50 Inhibidora , Riñón/citología , Kringles , Luz , Ratones , Modelos Biológicos , Mutación , Oligopéptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes de Fusión/metabolismo , Dispersión de Radiación , Difracción de Rayos XRESUMEN
RAPL, a protein that binds the small GTPase Rap1, is required for efficient immune cell trafficking. Here we have identified the kinase Mst1 as a critical effector of RAPL. RAPL regulated the localization and kinase activity of Mst1. 'Knockdown' of the gene encoding Mst1 demonstrated its requirement for the induction of both a polarized morphology and integrin LFA-1 clustering and adhesion triggered by chemokines and T cell receptor ligation. RAPL and Mst1 localized to vesicular compartments and dynamically translocated with LFA-1 to the leading edge upon Rap1 activation, suggesting a regulatory function for the RAPL-Mst1 complex in intracellular transport of LFA-1. Our study demonstrates a previously unknown function for Mst1 of relaying the Rap1-RAPL signal to induce cell polarity and adhesion of lymphocytes.
Asunto(s)
Adhesión Celular , Polaridad Celular , Factor de Crecimiento de Hepatocito/metabolismo , Linfocitos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Adhesión Celular/genética , Polaridad Celular/genética , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/análisis , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/química , Linfocitos/ultraestructura , Ratones , Ratones Noqueados , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/análisis , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismo , Regulación hacia Arriba , Proteínas de Unión al GTP rap1/agonistas , Proteínas de Unión al GTP rap1/análisis , Proteínas de Unión al GTP rap1/genéticaRESUMEN
An HGF antagonist, NK4, inhibits not only invasion and metastasis of tumor cells driven by HGF-Met receptor binding, but also tumor angiogenesis. To address the antitumor activities of NK4, we investigated the biological behaviors of CT26 transfected with the NK4 gene (CT26-NK4) in vitro and in vivo. In the in vitro assay, the invasion in MOCK transfected cells (control) was stimulated by HGF; however, in CT26-NK4 cells, these effects were completely inhibited. In the in vivo assay, the tumor growth of CT26-NK4 was strongly suppressed and the survival of CT26-NK4 tumor-bearing mice was significantly prolonged. Immunohistochemical analysis revealed that while proliferating cells (PCNA immunostaining) of CT26-NK4 tumors were weakly suppressed, the micro-vessel number (CD31/PECAM-1 immunostaining) in those tumors was significantly suppressed as compared with the control tumors. In conclusion, NK4 exerts potent antitumor effects via anti-angiogenesis rather than inhibition of biological events of tumor cells stimulated by HGF.
Asunto(s)
Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/genética , Mitógenos , Inhibidores de la Angiogénesis , Animales , Ratones , Neoplasias Experimentales/patología , Antígeno Nuclear de Célula en Proliferación/análisis , Transfección , Células Tumorales CultivadasRESUMEN
Hepatocyte growth factor (HGF), otherwise known as scatter factor (SF), has been demonstrated over the past decade to elicit a number of functions that may be tumorigenic, and enhance the invasive/metastatic nature of cancer cells. Clinical studies have also demonstrated that HGF/SF, together with its receptor, cMET, is closely related to the disease progression and prognosis of patients with cancer. The past few years have seen the identification of numerous inhibitors and antagonists to the action of HGF/SF. These factors have demonstrated a possible role in minimising the action of HGF/SF on cancer cells, and may be of therapeutic value in the future. This article overviews the activators, inhibitors, and antagonists to HGF/SF and discusses the possible implications in cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Serina Endopeptidasas/farmacología , Animales , Antineoplásicos/uso terapéutico , Biotransformación/efectos de los fármacos , Humanos , Neoplasias Hepáticas/patología , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/fisiologíaRESUMEN
The present study has investigated the functional role of the Met receptor in primary cultures of 20 papillary carcinomas and of normal thyroid cells obtained from the same patients. Normal and tumour cells grew as adherent cells, formed a confluent monolayer after 10-20 days, had epithelial morphology, and were immunoreactive for cytokeratin, vimentin, and thyroglobulin. The potential effect of hepatocyte growth factor (HGF) on cell invasiveness was investigated in Boyden chambers, using a nucleopore filter coated with Matrigel as the barrier and HGF as the chemoattractant. Tumour cells of five out of seven cases of papillary carcinoma were more responsive to HGF than the corresponding normal cells in terms of the number of migrated cells per mm(2). Involvement of the Met receptor in the HGF-induced migratory response was suggested by the observation that the agonistic anti-Met monoclonal antibody (MAb) DO-24 was equally effective. HGF did not affect the proliferative activity of thyroid cells. Under the same experimental conditions, 10 per cent fetal bovine serum (FBS) induced a two-fold increase in [(3)H]thymidine incorporation into normal cells and tumour cells. These findings are consistent with the possibility that HGF plays a crucial role in determining the invasiveness of tumour cells in papillary carcinoma of the thyroid.
Asunto(s)
Carcinoma Papilar/patología , Factor de Crecimiento de Hepatocito/farmacología , Neoplasias de la Tiroides/patología , Anticuerpos Monoclonales/farmacología , Western Blotting , División Celular/efectos de los fármacos , Técnicas de Cocultivo , Factor de Crecimiento de Hepatocito/agonistas , Humanos , Invasividad Neoplásica , Fosforilación , Proteínas Proto-Oncogénicas c-met/inmunología , Proteínas Proto-Oncogénicas c-met/metabolismo , Estimulación Química , Glándula TiroidesRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.
Asunto(s)
Empalme Alternativo , Factor de Crecimiento de Hepatocito/agonistas , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Expresión Génica , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento de Hepatocito/genética , Humanos , Riñón/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neoplasias/etiología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF is structurally related to plasminogen, including an amino-terminal hairpin loop, four kringle domains, and a serine protease-like region. A truncated HGF/SF isoform, designated HGF/NK2, which extends through the second kringle domain and behaves as a competitive HGF/SF antagonist, was previously shown to be encoded by an alternative HGF/SF transcript. In this study, we describe a second naturally occurring HGF/SF variant, HGF/NK1, consisting of the HGF/SF amino-terminal sequence and first kringle domain. This product is encoded by a 2-kilobase alternative transcript containing intronic sequence that was contiguous with exon K1b. Analysis of baculovirus-expressed HGF/NK1 revealed that this isoform possesses the heparin binding properties of HGF/SF and modest mitogenic and scattering activity relative to HGF/SF. However, at a 40-fold molar excess, HGF/NK1 inhibited HGF/SF-dependent DNA synthesis. HGF/NK1 stimulated tyrosine phosphorylation of Met, and covalent affinity cross-linking demonstrated a direct HGF/NK1-receptor interaction. These findings establish that the HGF/SF gene encodes multiple alternative products, which include not only a mitogenic agonist (HGF/SF) and a pure antagonist (HGF/NK2) but also a molecule with partial agonist/antagonist properties.