RESUMEN
Siderophores are specialized molecules produced by bacteria and fungi to scavenge iron, a crucial nutrient for growth and metabolism. Catecholate-type siderophores are mainly produced by bacteria, while hydroxamates are mostly from fungi. This study investigates the capacity of nine hydroxamate-type siderophores from fungi and Streptomyces to facilitate iron acquisition by the human pathogen Pseudomonas aeruginosa. Growth assays under iron limitation and 55Fe incorporation tests showed that all nine siderophores promoted bacterial growth and iron transport. The study also aimed to identify the TonB-dependent transporters (TBDTs) involved in iron import by these siderophores. Using mutant strains lacking specific TBDT genes, it was found that iron is imported into P. aeruginosa cells by FpvB for coprogen, triacetylfusarinine, fusigen, ferrirhodin, and ferrirubin. Iron complexed by desferioxamine G is transported by FpvB and FoxA, ferricrocin-Fe and ferrichrycin-Fe by FpvB and FiuA, and rhodotoluric acid-Fe by FpvB, FiuA, and another unidentified TBDT. These findings highlight the effectiveness of hydroxamate-type siderophores in iron transport into P. aeruginosa and provide insights into the complex molecular mechanisms involved, which are important for understanding microbial interactions and ecological balance.
Asunto(s)
Proteínas Bacterianas , Ácidos Hidroxámicos , Hierro , Pseudomonas aeruginosa , Sideróforos , Sideróforos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Hierro/metabolismo , Ácidos Hidroxámicos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Ferricromo/metabolismo , Ferricromo/análogos & derivados , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de la Membrana Bacteriana Externa , Proteínas de la Membrana , Receptores de Superficie CelularRESUMEN
Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.
RESUMEN
Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germlines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO/PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO/PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana. We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Proteína-Arginina N-Metiltransferasas , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Eucariontes/metabolismo , Plantas/metabolismo , Interferencia de ARN , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei. METHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics. FINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent. INTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings. FUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.
Asunto(s)
Colicinas , Shigella , Humanos , Colicinas/genética , Escherichia coli/genética , Shigella sonnei/genética , Estudio de Asociación del Genoma CompletoRESUMEN
Iron is an essential nutrient for the survival and virulence of Pseudomonas aeruginosa. The pathogen expresses at least 15 different iron-uptake pathways, the majority involving small iron chelators called siderophores. P. aeruginosa produces two siderophores, but can also use many produced by other microorganisms. This implies that the bacterium expresses appropriate TonB-dependent transporters (TBDTs) at the outer membrane to import the ferric form of each of the siderophores used. Here, we show that the two α-carboxylate-type siderophores rhizoferrin-Fe and staphyloferrin A-Fe are transported into P. aeruginosa cells by the TBDT ActA. Among the mixed α-carboxylate/hydroxamate-type siderophores, we found aerobactin-Fe to be transported by ChtA and schizokinen-Fe and arthrobactin-Fe by ChtA and another unidentified TBDT. Our findings enhance the understanding of the adaptability of P. aeruginosa and hold significant implications for developing novel strategies to combat antibiotic resistance.
Asunto(s)
Pseudomonas aeruginosa , Sideróforos , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Hierro/metabolismoRESUMEN
As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.
Asunto(s)
Nucleósidos , Espectrometría de Masas en Tándem , Animales , Bovinos , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Nucleósidos/análisis , Motor de Búsqueda , ARN de TransferenciaRESUMEN
Immunoprecipitation mass spectrometry (IP-MS) is a popular method for the identification of protein-protein interactions. This approach is particularly powerful when information is collected without a priori knowledge and has been successively used as a first key step for the elucidation of many complex protein networks. IP-MS consists in the affinity purification of a protein of interest and of its interacting proteins followed by protein identification and quantification by mass spectrometry analysis. We developed an R package, named IPinquiry, dedicated to IP-MS analysis and based on the spectral count quantification method. The main purpose of this package is to provide a simple R pipeline with a limited number of processing steps to facilitate data exploration for biologists. This package allows to perform differential analysis of protein accumulation between two groups of IP experiments, to retrieve protein annotations, to export results, and to create different types of graphics. Here we describe the step-by-step procedure for an interactome analysis using IPinquiry from data loading to result export and plot production.
Asunto(s)
Visualización de Datos , Proteínas , Proteínas/análisis , Espectrometría de Masas/métodos , Inmunoprecipitación , Cromatografía de AfinidadRESUMEN
Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.
RESUMEN
Metformin is one of the most prescribed antidiabetic agents worldwide and is also considered for other therapeutic applications including cancer and endocrine disorders. It is largely unmetabolized by human enzymes and its presence in the environment has raised concern, with reported toxic effects on aquatic life and potentially also on humans. We report on the isolation and characterisation of strain MD1, an aerobic methylotrophic bacterium growing with metformin as its sole carbon, nitrogen and energy source. Strain MD1 degrades metformin into dimethylamine used for growth, and guanylurea as a side-product. Sequence analysis of its fully assembled genome showed its affiliation to Aminobacter niigataensis. Differential proteomics and transcriptomics, as well as mini-transposon mutagenesis of the strain, point to genes and proteins essential for growth with metformin and potentially associated with hydrolytic C-N cleavage of metformin or with cellular transport of metformin and guanylurea. The obtained results suggest the recent evolution of the growth-supporting capacity of strain MD1 to degrade metformin. Our results identify candidate proteins of the enzymatic system for metformin transformation in strain MD1 and will inform future research on the fate of metformin and its degradation products in the environment and in humans.
RESUMEN
The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N',Nâ³-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.
Asunto(s)
Pseudomonas aeruginosa , Sideróforos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Benzamidas , Catecoles/metabolismo , Catecoles/farmacología , Bacterias Gramnegativas/metabolismo , Hidroxibenzoatos , Hierro/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismo , Sideróforos/farmacología , Zinc/metabolismoRESUMEN
RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Proteínas F-Box , MicroARNs , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , MicroARNs/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Iron is an essential nutriment for almost all organisms, but this metal is poorly bioavailable. During infection, bacteria access iron from the host by importing either iron or heme. Pseudomonas aeruginosa, a gram-negative pathogen, secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), to access iron and is also able to use many siderophores produced by other microorganisms (called xenosiderophores). To access heme, P. aeruginosa uses three distinct uptake pathways, named Has, Phu, and Hxu. We previously showed that P. aeruginosa expresses the Has and Phu heme uptake systems and the PVD- and PCH-dependent iron uptake pathways in iron-restricted growth conditions, using proteomic and RT-qPCR approaches. Here, using the same approaches, we show that physiological concentrations of hemin in the bacterial growth medium result in the repression of the expression of the proteins of the PVD- and PCH-dependent iron uptake pathways, leading to less production of these two siderophores. This indicates that the pathogen adapts its phenotype to use hemin as an iron source rather than produce PVD and PCH to access iron. Moreover, the presence of both hemin and a xenosiderophore resulted in (i) the strong induction of the expression of the proteins of the added xenosiderophore uptake pathway, (ii) repression of the PVD- and PCH-dependent iron uptake pathways, and (iii) no effect on the expression levels of the Has, Phu, or Hxu systems, indicating that bacteria use both xenosiderophores and heme to access iron.
Asunto(s)
Hierro , Pseudomonas aeruginosa , Hemina , Proteómica , SideróforosRESUMEN
In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Estabilidad del ARN , ARN de Planta/metabolismo , Dominio CatalíticoRESUMEN
Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, 55 Fe uptake assays, and LC-MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.
Asunto(s)
Pseudomonas aeruginosa , Sideróforos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cromatografía Liquida , Hierro/metabolismo , Fenoles , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Receptores de Superficie Celular/metabolismo , Sideróforos/metabolismo , Espectrometría de Masas en Tándem , TiazolesRESUMEN
Iron is an essential nutrient for bacterial growth and the cause of a fierce battle between the pathogen and host during infection. Bacteria have developed several strategies to access iron from the host, the most common being the production of siderophores, small iron-chelating molecules secreted into the bacterial environment. The opportunist pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a wide panoply of xenosiderophores, siderophores produced by other microorganisms. Here, we demonstrate that catecholamine neurotransmitters (dopamine, l-DOPA, epinephrine and norepinephrine) are able to chelate iron and efficiently bring iron into P. aeruginosa cells via TonB-dependent transporters (TBDTs). Bacterial growth assays under strong iron-restricted conditions and with numerous mutants showed that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake than for norepinephrine, epinephrine and l-DOPA, whereas PirA specificity appeared to be higher for l-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of l-DOPA.
Asunto(s)
Pseudomonas aeruginosa , Sideróforos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catecolaminas/metabolismo , Hierro/metabolismo , Neurotransmisores/metabolismo , Proteómica , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismoRESUMEN
Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Mitocondrias/metabolismo , ARN/metabolismo , Ribosomas/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestructura , Microscopía por Crioelectrón , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Ribosomas Mitocondriales/química , Ribosomas Mitocondriales/metabolismo , Ribosomas Mitocondriales/ultraestructura , ARN/química , ARN/genética , ARN/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/química , Ribosomas/genética , Ribosomas/ultraestructuraRESUMEN
The brain plays a key role in energy homeostasis, detecting nutrients, metabolites and circulating hormones from peripheral organs and integrating this information to control food intake and energy expenditure. Here, we show that a group of neurons in the Drosophila larval brain expresses the adiponectin receptor (AdipoR) and controls systemic growth and metabolism through insulin signaling. We identify glucose-regulated protein 78 (Grp78) as a circulating antagonist of AdipoR function produced by fat cells in response to dietary sugar. We further show that central AdipoR signaling inhibits peripheral Juvenile Hormone (JH) response, promoting insulin signaling. In conclusion, we identify a neuroendocrine axis whereby AdipoR-positive neurons control systemic insulin response.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Insulina/metabolismo , Neuronas/metabolismo , Receptores de Adiponectina/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Metabolismo Energético/genética , Hemolinfa/metabolismo , Homeostasis , Hormonas Juveniles/metabolismo , Larva/genética , Larva/metabolismo , Receptores de Adiponectina/genética , Transducción de Señal/genéticaRESUMEN
Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Interacciones Huésped-Patógeno , Defensa de la Planta contra la Herbivoria/genética , Virus de Plantas/fisiología , ARN Bicatenario/genética , ARN de Planta/genética , Proteínas de Unión al ARN/genética , Arabidopsis/virología , Proteínas de Arabidopsis/metabolismo , ARN Bicatenario/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación ViralRESUMEN
Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.
Asunto(s)
Alérgenos/química , Epidermis/química , Anhídridos Ftálicos/química , Proteínas/análisis , Isótopos de Carbono/química , Cromatografía Liquida , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteómica , Espectrometría de Masas en TándemRESUMEN
DNA polymerase η (pol η) is specifically required for translesion DNA synthesis across UV-induced DNA lesions. Recruitment of this error-prone DNA polymerase is tightly regulated during replication to avoid mutagenesis and perturbation of fork progression. Here, we report that pol η interacts with the calpain small subunit-1 (CAPNS1) in a yeast two-hybrid screening. This interaction is functional, as demonstrated by the ability of endogenous calpain to mediate calcium-dependent cleavage of pol η in cell-free extracts and in living cells treated with a calcium ionophore. The proteolysis of pol η was found to occur at position 465, leading to a catalytically active truncated protein containing the PCNA-interacting motif PIP1. Unexpectedly, cell treatment with the specific calpain inhibitor calpeptin resulted in a decreased extent of pol η foci after UV irradiation, indicating that calpain positively regulates pol η accumulation in replication foci.