RESUMO
How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.
Assuntos
Arabidopsis/efeitos dos fármacos , Dipeptídeos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Nicotiana/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulação por Computador , Dipeptídeos/química , Dipeptídeos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Plântula/efeitos dos fármacos , Plântula/metabolismo , Nicotiana/metabolismoRESUMO
An increasing number of drugs introduced to the market and numerous repositories of compounds with confirmed activity have posed the need to revalidate the state-of-the-art rules that determine the ranges of properties the compounds should possess to become future drugs. In this study, we designed a series of two chemotypes of aryl-piperazine hydantoin ligands of 5-HT7R, an attractive target in search for innovative CNS drugs, with higher molecular weight (close to or over 500). Consequently, 14 new compounds were synthesised and screened for their receptor activity accompanied by extensive docking studies to evaluate the observed structure-activity/properties relationships. The ADMET characterisation in terms of the biological membrane permeability, metabolic stability, hepatotoxicity, cardiotoxicity, and protein plasma binding of the obtained compounds was carried out in vitro. The outcome of these studies constituted the basis for the comprehensive challenge of computational tools for ADMET properties prediction. All the compounds possessed high affinity to the 5-HT7R (Ki below 250 nM for all analysed structures) with good selectivity over 5-HT6R and varying affinity towards 5-HT2AR, 5-HT1AR and D2R. For the best compounds of this study, the expression profile of genes associated with neurodegeneration, anti-oxidant response and anti-inflammatory function was determined, and the survival of the cells (SH-SY5Y as an in vitro model of Alzheimer's disease) was evaluated. One 5-HT7R agent (32) was characterised by a very promising ADMET profile, i.e. good membrane permeability, low hepatotoxicity and cardiotoxicity, and high metabolic stability with the simultaneous high rate of plasma protein binding and high selectivity over other GPCRs considered, together with satisfying gene expression profile modulations and neural cell survival. Such encouraging properties make it a good candidate for further testing and optimisation as a potential agent in the treatment of CNS-related disorders.
RESUMO
The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Glucosinolatos/metabolismo , HumanosRESUMO
Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Sementes/metabolismo , Sulfatos/metabolismo , Proteínas de Arabidopsis/metabolismo , Sementes/químicaRESUMO
Plants can mitigate environmental stress conditions through acclimation. In the case of fluctuating stress conditions such as high temperatures, maintaining a stress memory enables a more efficient response upon recurring stress. In a genetic screen for Arabidopsis thaliana mutants impaired in the memory of heat stress (HS) we have isolated the FORGETTER2 (FGT2) gene, which encodes a type 2C protein phosphatase (PP2C) of the D-clade. Fgt2 mutants acquire thermotolerance normally; however, they are defective in the memory of HS. FGT2 interacts with phospholipase D α2 (PLDα2), which is involved in the metabolism of membrane phospholipids and is also required for HS memory. In summary, we have uncovered a previously unknown component of HS memory and identified the FGT2 protein phosphatase and PLDα2 as crucial players, suggesting that phosphatidic acid-dependent signaling or membrane composition dynamics underlie HS memory.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Resposta ao Choque Térmico/fisiologia , Fosfolipase D/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Resposta ao Choque Térmico/genética , Fosfolipase D/fisiologia , Fosfoproteínas Fosfatases/genéticaRESUMO
During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Germinação , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Plântula/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Temperatura AltaRESUMO
Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.
Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteoma/análise , Proteínas Ribossômicas/genética , Ribossomos/genéticaRESUMO
The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.
Assuntos
Proteínas de Transporte/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Multimerização Proteica , Centrifugação com Gradiente de Concentração , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Deleção de GenesRESUMO
Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-ï¢-D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-ï¢-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-ï¢ -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.
Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Escherichia coli/efeitos dos fármacos , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tubercidina/análogos & derivados , Tubercidina/farmacologia , 2-Aminopurina/síntese química , Acetaldeído/análogos & derivados , Acetaldeído/química , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/enzimologia , Pirimidinas/química , Tubercidina/síntese químicaRESUMO
Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the l-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the l-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production.IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.
Assuntos
Coenzimas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Metaloproteínas/biossíntese , Proteínas de Escherichia coli/genética , Cofatores de Molibdênio , Proteômica , PteridinasRESUMO
Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein-Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein-small molecule and of protein-protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein-protein (PPIs) and protein-metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr-Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatografia em Gel/métodos , Metaboloma , Proteoma/metabolismo , Proteômica/métodos , Software , Ligação Proteica , Proteoma/isolamento & purificaçãoRESUMO
Stress granules (SGs) are evolutionary conserved aggregates of proteins and untranslated mRNAs formed in response to stress. Despite their importance for stress adaptation, no complete proteome composition has been reported for plant SGs. In this study, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs. To isolate SGs we used Arabidopsis seedlings expressing green fluorescent protein (GFP) fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification (AP). SGs isolates were analysed using mass spectrometry-based proteomics and metabolomics. A quarter of the identified proteins constituted known or predicted SG components. Intriguingly, the remaining proteins were enriched in key enzymes and regulators, such as cyclin-dependent kinase A (CDKA), that mediate plant responses to stress. In addition to proteins, nucleotides, amino acids and phospholipids also accumulated in SGs. Taken together, our results indicated the presence of a preexisting SG protein interaction network; an evolutionary conservation of the proteins involved in SG assembly and dynamics; an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Grânulos Citoplasmáticos/metabolismo , Metaboloma , Estresse Fisiológico , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteoma/metabolismo , Plântula/metabolismoRESUMO
2',3'-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2',3'-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2',3'-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2',3'-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2',3'-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2',3'-cAMP. Furthermore, SG formation was promoted in 2',3'-cAMP-treated Arabidopsis seedlings, and interactions between 2',3'-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2',3'-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.
Assuntos
Nucleotídeos de Adenina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Estresse Fisiológico , Proteínas de Arabidopsis/química , Cromatografia de Afinidade , Modelos Biológicos , Proteínas de Ligação a Poli(A)/química , Ligação Proteica , Domínios ProteicosRESUMO
Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9-ï¢-d- and N7-ï¢-d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6-ï¢-d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.
Assuntos
Guanina/química , Guanosina/química , Nucleosídeos/química , Purina-Núcleosídeo Fosforilase/química , Adenosina , Cinética , Nucleosídeos/análogos & derivados , Análise Espectral , Especificidade por SubstratoRESUMO
Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.
Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/química , Metalochaperonas/metabolismo , Níquel/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Teoria da Densidade Funcional , Escherichia coli/genética , Glicina/genética , Concentração de Íons de Hidrogênio , Metalochaperonas/química , Metalochaperonas/genética , Modelos Químicos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Níquel/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Zinco/química , Zinco/metabolismoRESUMO
A method for five-dimensional spectral reconstruction of non-uniformly sampled NMR data sets is proposed. It is derived from the previously published signal separation algorithm, with major alterations to avoid unfeasible processing of an entire five-dimensional spectrum. The proposed method allows credible reconstruction of spectra from as little as a few hundred data points and enables sensitive resonance detection in experiments with a high dynamic range of peak intensities. The efficiency of the method is demonstrated on two high-resolution spectra for rapid sequential assignment of intrinsically disordered proteins, namely 5D HN(CA)CONH and 5D (HACA)CON(CO)CONH.
Assuntos
Algoritmos , Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono , Humanos , Isótopos de Nitrogênio , Razão Sinal-Ruído , alfa-Sinucleína/químicaRESUMO
Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos de DNA Transponíveis , Inativação Gênica , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sítios de Ligação , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/metabolismo , Filogenia , Plantas Geneticamente Modificadas , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Tubulina (Proteína)/metabolismo , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteômica/métodosRESUMO
New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Isoformas de Proteínas , alfa-Sinucleína/química , Proteínas tau/químicaRESUMO
The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.