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BACKGROUND: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood. MATERIALS AND METHODS: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre- and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of metastatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes. RESULTS: We identified cell clusters associated with disease control or progression, defined differential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell subsets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abundance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort. CONCLUSIONS: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we support pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.
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Leucocitos Mononucleares , Melanoma , Humanos , Melanoma/patología , Linfocitos B , Transcriptoma , Estudios de Cohortes , InmunoterapiaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Although small cell lung cancer (SCLC) is treated as a homogeneous disease, biopsies and preclinical models reveal heterogeneity in transcriptomes and morphology. SCLC subtypes were recently defined by neuroendocrine transcription factor (NETF) expression. Circulating-tumor-cell-derived explant models (CDX) recapitulate donor patients' tumor morphology, diagnostic NE marker expression and chemotherapy responses. We describe a biobank of 38 CDX models, including six CDX pairs generated pretreatment and at disease progression revealing complex intra- and intertumoral heterogeneity. Transcriptomic analysis confirmed three of four previously described subtypes based on ASCL1, NEUROD1 and POU2F3 expression and identified a previously unreported subtype based on another NETF, ATOH1. We document evolution during disease progression exemplified by altered MYC and NOTCH gene expression, increased 'variant' cell morphology, and metastasis without strong evidence of epithelial to mesenchymal transition. This CDX biobank provides a research resource to facilitate SCLC personalized medicine.
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Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Bancos de Muestras Biológicas , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células Pequeñas/genéticaRESUMEN
Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Recurrencia Local de Neoplasia/diagnóstico , Células Neoplásicas Circulantes/patología , Venas Pulmonares/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/cirugía , Estadificación de NeoplasiasRESUMEN
Patient-derived xenograft (PDX) and circulating tumor cell-derived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNA- and RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX- and CDX-derived whole-exome and RNA-Seq data.Implications: This study describes a sensitive method to identify contaminating host reads in xenograft and explant DNA- and RNA-Seq data and is applicable to other forms of deep sequencing. Mol Cancer Res; 15(8); 1012-6. ©2017 AACR.
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Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias/genética , Células Neoplásicas Circulantes , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Algoritmos , Animales , Modelos Animales de Enfermedad , Exoma , Perfilación de la Expresión Génica , Humanos , Programas InformáticosRESUMEN
Clinical reading centers provide expertise for consistent, centralized analysis of medical data gathered in a distributed context. Accordingly, appropriate software solutions are required for the involved communication and data management processes. In this work, an analysis of general requirements and essential architectural and software design considerations for reading center information systems is provided. The identified patterns have been applied to the implementation of the reading center platform which is currently operated at the Center of Ophthalmology of the University Hospital of Tübingen.
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Investigación Biomédica , Sistemas de Registros Médicos Computarizados , Diseño de Software , Programas Informáticos , Humanos , Aplicaciones de la Informática Médica , Registro Médico CoordinadoRESUMEN
PURPOSE: Abnormalities of blood vessel anatomy, morphology, and ratio can serve as important diagnostic markers for retinal diseases such as AMD or diabetic retinopathy. Large cohort studies demand automated and quantitative image analysis of vascular abnormalities. Therefore, we developed an analytical software tool to enable automated standardized classification of blood vessels supporting clinical reading. METHODS: A dataset of 61 images was collected from a total of 33 women and 8 men with a median age of 38 years. The pupils were not dilated, and images were taken after dark adaption. In contrast to current methods in which classification is based on vessel profile intensity averages, and similar to human vision, local color contrast was chosen as a discriminator to allow artery vein discrimination and arterial-venous ratio (AVR) calculation without vessel tracking. RESULTS: With 83% ± 1 standard error of the mean for our dataset, we achieved best classification for weighted lightness information from a combination of the red, green, and blue channels. Tested on an independent dataset, our method reached 89% correct classification, which, when benchmarked against conventional ophthalmologic classification, shows significantly improved classification scores. CONCLUSIONS: Our study demonstrates that vessel classification based on local color contrast can cope with inter- or intraimage lightness variability and allows consistent AVR calculation. We offer an open-source implementation of this method upon request, which can be integrated into existing tool sets and applied to general diagnostic exams.
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Algoritmos , Clasificación/métodos , Interpretación de Imagen Asistida por Computador/métodos , Arteria Retiniana/anatomía & histología , Enfermedades de la Retina/diagnóstico , Vena Retiniana/anatomía & histología , Adulto , Color , Adaptación a la Oscuridad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Arteria Retiniana/citología , Arteria Retiniana/patología , Enfermedades de la Retina/patología , Vena Retiniana/citología , Vena Retiniana/patología , Programas InformáticosRESUMEN
Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Fosfatos de Inositol/metabolismo , Oxilipinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiologíaRESUMEN
Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Membrana Celular/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferencia de Fosfolípidos/fisiología , Raíces de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Sitios de Unión , Polaridad Celular , Simulación de Dinámica Molecular , Morfogénesis , Proteínas de Transferencia de Fosfolípidos/química , Raíces de Plantas/crecimiento & desarrollo , Unión Proteica , Transducción de SeñalRESUMEN
In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH4(+), NH3 + H(+), NH4(+) + H(+) or NH3, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH3 transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH3 across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH3 transport through the central subunit pore is suggested.
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Amoníaco/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Transporte Iónico , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Especificidad por SustratoRESUMEN
Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These host-defense peptides compromise the integrity of microbial cell membranes and thus evade pathways by which bacteria develop rapid antibiotic resistance. Although more than 1,700 host-defense peptides have been identified, the structural and mechanistic basis of their action remains speculative. This impedes the desired rational development of these agents into next-generation antibiotics. We present the X-ray crystal structure as well as solid-state NMR spectroscopy, electrophysiology, and MD simulations of human dermcidin in membranes that reveal the antibiotic mechanism of this major human antimicrobial, found to suppress Staphylococcus aureus growth on the epidermal surface. Dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidate the unusual membrane permeation pathway for ions and show adjustment of the pore to various membranes. Our study unravels the comprehensive mechanism for the membrane-disruptive action of this mammalian host-defense peptide at atomistic level. The results may form a foundation for the structure-based design of peptide antibiotics.
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Antiinfecciosos/química , Antiinfecciosos/farmacología , Canales Iónicos/química , Canales Iónicos/farmacología , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Péptidos/química , Péptidos/farmacología , Cristalografía por Rayos X , Humanos , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.
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Alanina , Proteínas de Arabidopsis/química , Arabidopsis/citología , Nucléolo Celular/metabolismo , Proteínas de Unión al ADN/química , Multimerización de Proteína , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/metabolismo , Biología Computacional , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Filogenia , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Homología de Secuencia de Aminoácido , Electricidad Estática , Factores de Transcripción/químicaRESUMEN
Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.
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Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Evolución Molecular Dirigida , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fenotipo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Conformación Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Transducción de Señal , Red trans-Golgi/metabolismoRESUMEN
Phosphoinositides, phosphorylated species of phosphatidylinositol (PtdIns), are critical regulatory lipids in all eukaryotic cells. The molecular mechanisms that lead to the phosphorylation of an individual PtdIns- or phosphoinositide molecule remain largely unkown even though lipid kinases and phosphatases involved in these processes have been studied in detail. The observation by us and others that liposomal PtdIns (and phosphoinositide) molecules are poor in vitro substrates for kinases and phosphatases raises the question of how these enzymes execute their function in living cells. Recent work indicates that Sec14, the founding member of a large superfamily of eukaryotic proteins, is crucial for the process of PtdIns phosphorylation. The collective data suggest that Sec14 mediates a heterotypic phospholipid exchange reaction of PtdIns with phosphatidylcholine (PtdCho) during which PtdIns becomes vulnerable for kinase attack and thereby promotes the generation of phosphoinositides.1,2 In a recent paper we address the molecular mechanism of this phospholipid (PL) exchange reaction in a pseudo-Sec14 protein (Sfh1) that we rendered functional by a directed evolution approach. We find that enhanced PL-cycling into and out of the hydrophobic pocket of these activated Sfh1 mutants depends on the reconfiguration of interactions between a C-terminal string motif and the floor of the hydrophobic pocket that results in increased oscillations in a helical gate that controls pocket access. Here we further discuss our findings and propose molecular dynamics simulations as a tool to approach energetically unfavorable transition states and to identify novel protein-ligand interactions invisible to X-ray crystallography.
RESUMEN
Nitrate is frequently the major nitrogen source for plants and is generally assimilated during the day. In the absence of light, nitrate can transiently accumulate in the vacuolar lumen via tonoplast transporters. CLC-a, a member of the CLC family of anion transporters, is critically involved in this nitrate storage in the vacuole, while other CLC family members apparently have different roles in diverse cell organelles. Here, CLC-b, a close relative of CLC-a, was functionally expressed in oocytes and analyzed. CLC-b conducted strongly outwardly rectifying anionic currents that were largest in the presence of nitrate. Fluorescence ratio changes of oocytes loaded with a pH-dependent fluorescent dye suggested that NO(-)(3) transport is associated with H(+) exchange. CLC-b was localized at the tonoplast, as was CLC-c, when tagged with the green fluorescent protein. CLC-b expression was strongest in young roots, hypocotyl and cotyledons. The physiological role of CLC-b was analyzed using two independent knock-out alleles. Both lines grew as the wild type in various conditions. The total chloride and nitrate content was identical in clcb lines and the wild type, potentially suggesting that mutants were able to compensate the loss of CLC-b.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales de Cloruro/metabolismo , Nitratos/metabolismo , Vacuolas/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Canales de Cloruro/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Insercional , Mutación , Oocitos , XenopusRESUMEN
Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that NH(4)(+) is the recruited substrate, but the uncharged form NH(3) is conducted. AtAMT2 partially co-localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Plantas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Animales , Arabidopsis/anatomía & histología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Modelos Moleculares , Oocitos/citología , Oocitos/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Compuestos de Amonio Cuaternario/química , Xenopus laevisRESUMEN
Plants contain a large number of aquaporins with different selectivity. These channels generally conduct water, but some additionally conduct NH(3), CO(2) and/or H(2)O(2). The experimental evidence and molecular basis for the transport of a given solute, the validation with molecular dynamics simulations and the physiological impact of the selectivity are reviewed here. The aromatic/arginine (ar/R) constriction is most important for solute selection, but the exact pore requirements for efficient conduction of small solutes remain difficult to predict. Yeast growth assays are valuable for screening substrate selectivity and are explicitly shown for hydrogen peroxide and methylamine, a transport analog of ammonia. Independent assays need to address the relevance of different substrates for each channel in its physiological context. This is emphasized by the fact that several plant NIP channels, which conduct several solutes, are specifically involved in the transport of metalloids, such as silicic acid, arsenite, or boric acid in planta.
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Acuaporinas/fisiología , Proteínas de Plantas/fisiología , Plantas/metabolismo , Amoníaco/metabolismo , Acuaporinas/química , Acuaporinas/metabolismo , Transporte Biológico , Dióxido de Carbono/metabolismo , Permeabilidad de la Membrana Celular , Simulación por Computador , Modelos Moleculares , Oxígeno/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , Protones , Transducción de Señal , Urea/metabolismo , Agua/metabolismoRESUMEN
In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH(4)(+) seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH(3) instead of NH(4)(+). Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH(3) into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus uptake.
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Proteínas de Transporte de Catión/fisiología , Glomeromycota/metabolismo , Lotus/metabolismo , Micorrizas/metabolismo , Nitrógeno/metabolismo , Proteínas de Plantas/fisiología , Animales , Transporte Biológico/fisiología , Proteínas de Transporte de Catión/química , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno , Cinética , Lotus/microbiología , Modelos Biológicos , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Saccharomyces cerevisiae/genética , Análisis de Secuencia de Proteína , Regulación hacia Arriba , XenopusRESUMEN
Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH(3), and a residue without direct contact to the pore lumen was critical for conduction in the mutants.