RESUMO
Extracellular free heme can intercalate into membranes and promote damage to cellular macromolecules. Thus it is likely that specific intercellular pathways exist for the directed transport, trafficking, and delivery of heme to cellular destinations, although none have been found to date. Here we show that Caenorhabditis elegans HRG-3 is required for the delivery of maternal heme to developing embryos. HRG-3 binds heme and is exclusively secreted by maternal intestinal cells into the interstitial fluid for transport of heme to extraintestinal cells, including oocytes. HRG-3 deficiency results either in death during embryogenesis or in developmental arrest immediately post-hatching-phenotypes that are fully suppressed by maternal but not zygotic hrg-3 expression. Our results establish a role for HRG-3 as an intercellular heme-trafficking protein.
Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Heme/deficiência , Hemeproteínas/química , Hemeproteínas/genética , Mucosa Intestinal/metabolismo , Mutação , Fenótipo , Transporte Proteico , Via SecretóriaRESUMO
Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consistent with a role in iron efflux, LIR1 deficiency does not affect iron uptake by L. amazonensis but significantly increases the amount of iron retained intracellularly in the parasites. LIR1 null parasites are more sensitive to iron toxicity and have drastically impaired infectivity, phenotypes that are reversed by LIR1 complementation. We conclude that LIR1 functions as a plasma membrane iron exporter with a critical role in maintaining iron homeostasis and promoting infectivity in L. amazonensis.
Assuntos
Membrana Celular/metabolismo , Ferro/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/prevenção & controle , Proteínas de Protozoários/metabolismo , Virulência/efeitos dos fármacos , Animais , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/parasitologia , Transporte Biológico , Células Cultivadas , Feminino , Homeostase , Ferro/toxicidade , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/genéticaRESUMO
The roundworm Caenorhabditis elegans is a heme auxotroph that requires the coordinated actions of HRG-1 heme permeases to transport environmental heme into the intestine and HRG-3, a secreted protein, to deliver intestinal heme to other tissues including the embryo. Here we show that heme homeostasis in the extraintestinal hypodermal tissue was facilitated by the transmembrane protein HRG-2. Systemic heme deficiency up-regulated hrg-2 mRNA expression over 200-fold in the main body hypodermal syncytium, hyp 7. HRG-2 is a type I membrane protein that binds heme and localizes to the endoplasmic reticulum and apical plasma membrane. Cytochrome heme profiles are aberrant in HRG-2-deficient worms, a phenotype that was partially suppressed by heme supplementation. A heme-deficient yeast strain, ectopically expressing worm HRG-2, revealed significantly improved growth at submicromolar concentrations of exogenous heme. Taken together, our results implicate HRG-2 as a facilitator of heme utilization in the Caenorhabditis elegans hypodermis and provide a mechanism for the regulation of heme homeostasis in an extraintestinal tissue.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Tela Subcutânea/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Hemeproteínas/química , Hemeproteínas/genética , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Growing evidence in vertebrates predicts that cellular haem levels in animals are maintained not only by a cell's internal capacity for haem synthesis in a cell-autonomous manner, but also by an inter-organ haem trafficking network through cell-non-autonomous regulation. Using Caenorhabditis elegans, a genetically and optically amenable animal model for visualizing haem-dependent signalling, we show that HRG-7, a protein with homology to aspartic proteases, mediates inter-organ signalling between the intestine and extra-intestinal tissues. Intestinal HRG-7 functions as a secreted signalling factor during haem starvation in extra-intestinal tissues and is regulated through a DBL-1, homologous to BMP5, dependent signal from neurons. Given the evidence that vertebrate homologues exist for each of the components of the HRG-7-mediated signalling pathway, it is conceivable that the cell-non-autonomous signalling framework that we uncovered in C. elegans may have functional relevance for inter-organ regulation of iron and haem metabolism in humans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais , Animais , Animais Geneticamente Modificados , Transporte Biológico , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Heme/deficiência , Hemeproteínas/genética , Homeostase , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Interferência de RNA , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.
Assuntos
Heme/metabolismo , Leishmania mexicana/patogenicidade , Proteínas de Protozoários/metabolismo , Tirosina , Sequência de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans/genética , Feminino , Genes Reporter , Humanos , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Macrófagos/parasitologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , VirulênciaRESUMO
In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Animais , Animais Geneticamente Modificados , Meios de Cultura , Heme , Tamanho da PartículaRESUMO
Adult humans have about 25 trillion red blood cells (RBCs), and each second we recycle about 5 million RBCs by erythrophagocytosis (EP) in macrophages of the reticuloendothelial system. Despite the central role for EP in mammalian iron metabolism, the molecules and pathways responsible for heme trafficking during EP remain unknown. Here, we show that the mammalian homolog of HRG1, a transmembrane heme permease in C. elegans, is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during EP. HRG1 is strongly expressed in macrophages of the reticuloendothelial system and specifically localizes to the phagolysosomal membranes during EP. Depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment. Importantly, missense polymorphisms in human HRG1 are defective in heme transport. Our results reveal HRG1 as the long-sought heme transporter for heme-iron recycling in macrophages and suggest that genetic variations in HRG1 could be modifiers of human iron metabolism.