RESUMEN
The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Venenos de Cnidarios/química , Helmintos/metabolismo , Memoria Inmunológica/efectos de los fármacos , Canal de Potasio Kv1.3/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Electrofisiología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Hipersensibilidad Tardía/prevención & control , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Filogenia , Conformación Proteica , Ratas , Ratas Endogámicas Lew , Receptores CCR7/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Haems are metalloporphyrins that serve as prosthetic groups for various biological processes including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control, metabolic reprogramming and microRNA processing. With a few exceptions, haem is synthesized by a multistep biosynthetic pathway comprising defined intermediates that are highly conserved throughout evolution. Despite our extensive knowledge of haem biosynthesis and degradation, the cellular pathways and molecules that mediate intracellular haem trafficking are unknown. The experimental setback in identifying haem trafficking pathways has been the inability to dissociate the highly regulated cellular synthesis and degradation of haem from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural haem auxotrophs that acquire environmental haem for incorporation into haemoproteins, which have vertebrate orthologues. Here we show, by exploiting this auxotrophy to identify HRG-1 proteins in C. elegans, that these proteins are essential for haem homeostasis and normal development in worms and vertebrates. Depletion of hrg-1, or its paralogue hrg-4, in worms results in the disruption of organismal haem sensing and an abnormal response to haem analogues. HRG-1 and HRG-4 are previously unknown transmembrane proteins, which reside in distinct intracellular compartments. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations and, most strikingly, profound defects in erythropoiesis-phenotypes that are fully rescued by worm HRG-1. Human and worm proteins localize together, and bind and transport haem, thus establishing an evolutionarily conserved function for HRG-1. These findings reveal conserved pathways for cellular haem trafficking in animals that define the model for eukaryotic haem transport. Thus, uncovering the mechanisms of haem transport in C. elegans may provide insights into human disorders of haem metabolism and reveal new drug targets for developing anthelminthics to combat worm infestations.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Hemo/metabolismo , Hemoproteínas/metabolismo , Homeostasis , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Eritropoyesis , Hemo/farmacología , Hemoproteínas/genética , Humanos , Metaloporfirinas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Obesity-related chronic low-grade inflammation plays a central role in the development of insulin resistance. Macrophages are key players in adipose tissue homeostasis, and their phenotypic shift from the anti-inflammatory or alternatively activated (M2) form to the pro-inflammatory, classically activated (M1) form is a hallmark of insulin resistance. However, adipose tissue macrophages (ATMs) have been identified as a distinct subpopulation of macrophages in several recent studies. These ATMs, described as metabolically activated macrophages (MMe), differ from M1 and are primarily found in the adipose tissue of obese individuals. In our study, we developed an in vitro model of MMe macrophages to establish a simple and reproducible system to understand their characteristics and role in the pathophysiology of insulin resistance. We examined their characteristics such as inflammatory patterns, surface markers, and metabolic features, and compared them with M1 and M2 macrophages. We found that a cell line-based in vitro model effectively mirrors the characteristics of ATMs, highlighting distinct inflammatory phenotypes, metabolism, surface markers, altered lysosomal activity, and ER stress akin to macrophages in vivo. This model captures the subtle distinctions between MMe and M1, and can be effectively used to study several features of macrophage-adipose interactions of therapeutic importance.
Asunto(s)
Tejido Adiposo , Inflamación , Resistencia a la Insulina , Macrófagos , Obesidad , Macrófagos/metabolismo , Tejido Adiposo/metabolismo , Humanos , Obesidad/metabolismo , Obesidad/patología , Inflamación/patología , Inflamación/metabolismo , Activación de Macrófagos , Animales , Estrés del Retículo Endoplásmico , RatonesRESUMEN
The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide's concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide's absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186's slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Canal de Potasio Kv1.3/antagonistas & inhibidores , Proteínas/farmacología , Linfocitos T/efectos de los fármacos , Absorción/efectos de los fármacos , Absorción/inmunología , Animales , Artritis/tratamiento farmacológico , Artritis/inmunología , Artritis/metabolismo , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Humanos , Concentración 50 Inhibidora , Canal de Potasio Kv1.3/inmunología , Canal de Potasio Kv1.3/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Macaca fascicularis , Bloqueadores de los Canales de Potasio/inmunología , Bloqueadores de los Canales de Potasio/farmacocinética , Bloqueadores de los Canales de Potasio/farmacología , Proteínas/farmacocinética , Ratas , Ratas Sprague-Dawley , Saimiri , Linfocitos T/inmunología , Linfocitos T/metabolismo , Distribución Tisular/efectos de los fármacos , Distribución Tisular/inmunologíaRESUMEN
Voltage-gated K(+) channels undergo a voltage-dependent conductance change that plays a key role in modulating cellular excitability. While the Open state is captured in crystal structures of Kv1.2 and a chimeric Kv1.2/Kv2.1 channel, the Close state and the mechanism of this transition are still a subject of debate. Here, we propose a model based on mutagenesis combined with measurements of both ionic and gating currents which is consistent with the idea that the Open state is the default state, the energy of the electric field being used to keep the channel closed. Our model incorporates an 'Activated state' where the bulk of sensor movement is completed without channel opening. The model accounts for the well characterized electrophysiology of the 'V2' and 'ILT' mutations in Shaker, where sensor movement and channel opening occur over distinct voltage ranges. Moreover, the model proposes relatively small protein rearrangements in going from the Activated to the Open state, consistent with the rapid transitions observed in single channel records of Shaker type channels at zero millivolts.
Asunto(s)
Activación del Canal Iónico/fisiología , Canal de Potasio Kv.1.2/química , Canal de Potasio Kv.1.2/fisiología , Modelos Biológicos , Secuencia de Aminoácidos , Animales , Cristalización , Femenino , Canal de Potasio Kv.1.2/genética , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Mutación/genética , Oocitos/citología , Oocitos/fisiología , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Electrophysiological and pharmacological studies coupled with molecular identification have revealed a unique network of ion channels--Kv1.3, KCa3.1, CRAC (Orai1 + Stim1), TRPM7, Cl(swell)--in lymphocytes that initiates and maintains the calcium signaling cascade required for activation. The expression pattern of these channels changes during lymphocyte activation and differentiation, allowing the functional network to adapt during an immune response. The Kv1.3 channel is of interest because it plays a critical role in subsets of T and B lymphocytes implicated in autoimmune disorders. The ShK toxin from the sea anemone Stichodactyla helianthus is a potent blocker of Kv1.3. ShK-186, a synthetic analog of ShK, is being developed as a therapeutic for autoimmune diseases, and is scheduled to begin first-in-man phase-1 trials in 2011. This review describes the journey that has led to the development of ShK-186.