RESUMO
Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.
Assuntos
Descoberta de Drogas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Large multifunctional peptidase 7 (LMP7/ß5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).
Assuntos
Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Compostos de Boro/administração & dosagem , Bortezomib/administração & dosagem , Proliferação de Células , Feminino , Glicina/administração & dosagem , Glicina/análogos & derivados , Humanos , Camundongos , Camundongos Nus , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteólise , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In tobacco, the heavy metal P1B-ATPases HMA4.1 and HMA4.2 function in root-to-shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root-to-shoot transfer by >90%. Analysis of plants with segregating null and wild-type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild-type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.
Assuntos
Cádmio/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Mutação/genética , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Nicotiana/genética , Zinco/metabolismoRESUMO
Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ácido Fítico/metabolismo , Transdução de Sinais/fisiologia , Adenilil Imidodifosfato/farmacologia , Proteínas de Arabidopsis/genética , Transporte Biológico/efeitos dos fármacos , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fosfatos de Inositol/metabolismo , Microscopia Confocal , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Fósforo/metabolismo , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/metabolismo , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.
Assuntos
Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Linhagem Celular , Genes Reporter , Glioma/metabolismo , Glioma/patologia , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Neurônios/citologia , Tomografia por Emissão de Pósitrons , Reprodutibilidade dos Testes , Transplante de Células-Tronco , Células-Tronco/citologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Targeted therapies directed against individual cancer-specific molecular alterations offer the development of disease-specific and individualized treatment strategies. Activation of the transcription factor E2F-1 via alteration of the p16-cyclinD-Rb pathway is one of the key molecular events in the development of gliomas. E2F-1 binds to and activates the E2F-1 promoter in an autoregulatory manner. The human E2F-1 promoter has been shown to be selectively activated in tumor cells with a defect in the pRb pathway. Paradoxically, E2F-1 also carries tumor suppressor function. Our investigations focused on analyzing the dynamics of the activity of the E2F-1 responsive element under basal conditions and certain stimuli such as chemotherapy using molecular imaging technology. We constructed a retrovirus bearing the Cis-E2F-TA-LITG reporter system to noninvasively assess E2F-1-dependent transcriptional regulation in culture and in vivo. We show that our reporter system is sensitive to monitor various changes in cellular E2F-1 levels and its transcriptional control of our reporter system to follow the state of the Rb/E2F pathway and the DNA damage-induced up-regulation of E2F-1 activity in vivo. Exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea leads to increased E2F-1 expression levels in a dose- and time-dependent manner, which can be quantified by imaging in vivo, leading to an alteration of cell cycle progression and caspase 3/7 activity. In summary, noninvasive imaging of E2F-1 as a common downstream regulator of cell cycle progression using the Cis-E2F-TA-LUC-IRES-TKGFP reporter system is highly attractive for evaluating the kinetics of cell cycle regulation and the effects of novel cell cycle targeting anticancer agents in vivo.
Assuntos
Neoplasias Encefálicas/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Microscopia de Fluorescência/métodos , Transcrição Gênica , Animais , Neoplasias Encefálicas/genética , Carmustina/farmacologia , Linhagem Celular Tumoral , Dano ao DNA , Progressão da Doença , Glioma/genética , Humanos , Camundongos , Camundongos Nus , Microscopia de Fluorescência/instrumentação , Retroviridae/genéticaRESUMO
The enormous metabolic plasticity of plants allows detoxification of many harmful compounds that are generated during biosynthetic processes or are present as biotic or abiotic toxins in their environment. Derivatives of toxic compounds such as glutathione conjugates are moved into the central vacuole via ATP-binding cassette (ABC)-type transporters of the multidrug resistance-associated protein (MRP) subfamily. The Arabidopsis genome contains 15 AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) cluster together in one of two major phylogenetic clades. We isolated T-DNA knockout alleles in all four highly homologous AtMRP genes of this clade and subjected them to physiological analysis to assess the function of each AtMRP of this group. None of the single atmrp mutants displayed visible phenotypes under control conditions. In spite of the fact that AtMRP1 and AtMRP2 had been described as efficient ATP-dependent organic anion transporters in heterologous expression experiments, the contribution of three of the AtMRP genes (1, 11 and 12) to detoxification is marginal. Only knockouts in AtMRP2 exhibited a reduced sensitivity towards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides. AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophyll degradation since ethylene-treated rosettes of atmrp2 showed reduced senescence, and AtMRP2 expression is induced during senescence. This suggests that AtMRP2 is involved in vacuolar transport of chlorophyll catabolites. Vacuolar uptake studies demonstrated that transport of typical MRP substrates was reduced in atmrp2. We conclude that within clade I, only AtMRP2 contributes significantly to overall organic anion pump activity in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Clorofila/metabolismo , Inativação Metabólica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/metabolismo , Vacúolos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Proteínas de Arabidopsis/genética , Transporte Biológico , Cotilédone/genética , Análise Mutacional de DNA , DNA Bacteriano , Flores/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosidases/metabolismo , Mutação/genética , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , Testes de ToxicidadeRESUMO
Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética , Glioma/genética , Glioma/terapia , Luminescência , Animais , Western Blotting , Neoplasias Encefálicas/metabolismo , Vetores Genéticos , Glioma/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 1/genética , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina Quinase/genética , Timidina Quinase/metabolismo , Células Tumorais CultivadasRESUMO
To further develop gene therapy for patients with glioblastomas, an experimental gene therapy protocol was established comprising a series of imaging parameters for (i) noninvasive assessment of viable target tissue followed by (ii) targeted application of herpes simplex virus type 1 (HSV-1) amplicon vectors and (iii) quantification of treatment effects by imaging. We show that viable target tissue amenable for application of gene therapy vectors can be identified by multitracer positron emission tomography (PET) using 2-(18)F-fluoro-2-deoxy-D-glucose, methyl-(11)C-L-methionine, or 3'-deoxy-3'-(18)F-fluoro-L-thymidine ([(18)F]FLT). Targeted application of HSV-1 amplicon vectors containing two therapeutic genes with synergistic antitumor activity (Escherichia coli cytosine deaminase, cd, and mutated HSV-1 thymidine kinase, tk39, fused to green fluorescent protein gene, gfp) leads to an overall response rate of 68%, with 18% complete responses and 50% partial responses. Most importantly, we show that the "tissue dose" of HSV-1 amplicon vector-mediated gene expression can be noninvasively assessed by 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) PET. Therapeutic effects could be monitored by PET with significant differences in [(18)F]FLT accumulation in all positive control tumors and 72% in vivo transduced tumors (P = 0.01) as early as 4 days after prodrug therapy. For all stably and in vivo transduced tumors, cdIREStk39gfp gene expression as measured by [(18)F]FHBG-PET correlated with therapeutic efficiency as measured by [(18)F]FLT-PET. These data indicate that imaging-guided vector application with determination of tissue dose of vector-mediated gene expression and correlation to induced therapeutic effect using multimodal imaging is feasible. This strategy will help in the development of safe and efficient gene therapy protocols for clinical application.
Assuntos
Terapia Genética/métodos , Glioma/diagnóstico por imagem , Glioma/terapia , Animais , Linhagem Celular Tumoral , Didesoxinucleosídeos , Radioisótopos de Flúor , Glioma/genética , Humanos , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Ratos , Ratos NusRESUMO
Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.
Assuntos
Ácido Abscísico/química , Trifosfato de Adenosina/química , Ânions , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Teste de Complementação Genética , Glibureto/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/química , Ligação Proteica , Protoplastos/metabolismo , Transdução de Sinais , Estomas CirúrgicosRESUMO
ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.
Assuntos
Membrana Celular/metabolismo , Desastres , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Arabidopsis/genética , Dióxido de Carbono/metabolismo , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes Reporter , Cinética , Luz , Metotrexato/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Transpiração Vegetal , Transporte Proteico , Células EstromaisRESUMO
The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes. Due to broad substrate spectra, specific modulators of these proteins are still lacking. Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor. Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants. To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated. Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein. Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide. Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist. Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização , Compostos de Sulfonilureia/farmacologia , Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacocinética , Proteínas Fúngicas/efeitos dos fármacos , Glucuronídeos/metabolismo , Glibureto/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Plantas/efeitos dos fármacos , Canais de Potássio , Receptores de Droga , Receptores de Sulfonilureias , Vacúolos/metabolismoRESUMO
Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Resistência a Múltiplos Medicamentos/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Família Multigênica/genética , Filogenia , Regiões Promotoras Genéticas/genética , Pseudogenes/genética , Regulação para Cima/genéticaRESUMO
IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Diferenciação Celular/genética , Deficiências de Ferro , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Proteínas de Transporte de Cátions/metabolismo , DNA Bacteriano/genética , Ferro/metabolismo , Mutagênese Insercional , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zinco/metabolismoRESUMO
Vacuolar sequestration or cellular extrusion of glutathione-conjugated xenobiotics and catabolites by ATP-binding cassette (ABC) transporters is an important detoxification mechanism operating in many species. In this study, we show that the yeast ABC transporter Bpt1p, a paralogue of Ycf1p, acts as an ATP-dependent vacuolar pump for glutathione conjugates. Bpt1p, which is inhibited by vanadate and glibenclamide, accounts for one third of the total vacuolar transport of glutathione conjugates. Furthermore, immunoblot analyses show that Bpt1p levels are strongly elevated in early stationary phase, consistent with a function of Bpt1p in vacuolar detoxification.