RESUMO
Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metanol/análogos & derivados , Compostos Organosselênicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Metanol/farmacologia , Chaperonas Moleculares/metabolismo , Oxirredução/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.
Assuntos
Metanol/análogos & derivados , Compostos Organosselênicos/metabolismo , Agregação Patológica de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Metanol/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Integrases/metabolismo , Espermidina/farmacologia , Sítios de Ligação , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Fatores Hospedeiros de Integração/metabolismoRESUMO
Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3Î-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Ligação Competitiva , Integrases/metabolismo , Sequências Repetidas Invertidas , Plasmídeos/metabolismo , Ligação ProteicaRESUMO
Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity.
Assuntos
Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Selenometionina/metabolismo , Selenometionina/toxicidade , Aminoácidos Sulfúricos/metabolismo , Aminoácidos Sulfúricos/toxicidade , Reparo do DNA , Suplementos Nutricionais/toxicidade , Humanos , Redes e Vias Metabólicas/genética , Metionina/metabolismo , Mutação , Estresse Oxidativo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Ácido Selenioso/metabolismo , Ácido Selenioso/toxicidade , Compostos de Selênio/metabolismo , Compostos de Selênio/toxicidade , Selenocisteína/análogos & derivados , Selenocisteína/metabolismoRESUMO
D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Proteínas de Escherichia coli/genética , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Estereoisomerismo , Termodinâmica , Tirosina-tRNA Ligase/genéticaRESUMO
It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution. Here, we analyze the non-electrostatic component of the free-energy difference between B-DNA and Z-DNA. We also compute the composition of the counterion sheath in a wide range of mixed-salt solutions and of temperatures: in contrast with the large difference of composition between the B-DNA and Z-DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed-salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small-Angle X-ray Scattering and complemented by (31) P NMR. With due consideration for the kinetics of the B-Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5'-d[T-(m(5) C-G)12 -T] 26-mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson-Boltzmann equation is described in detail. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 369-384, 2016.
Assuntos
DNA de Forma B/química , DNA Forma Z/química , Oligonucleotídeos/química , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Sais/química , Espalhamento a Baixo Ângulo , Soluções , Temperatura , Termodinâmica , Difração de Raios XRESUMO
In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.
Assuntos
Hidrolases de Éster Carboxílico/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , RNA Bacteriano/química , Aminoacil-RNA de Transferência/química , RNA de Transferência de Histidina/química , Sítios de Ligação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Catálise , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Mapeamento de Peptídeos , Estrutura Secundária de Proteína , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/metabolismoRESUMO
Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P(i) concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P(i) transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P(i) medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P(i) transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.
Assuntos
Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Selenito de Sódio/metabolismo , Meios de Cultura/química , Proteínas de Transporte de Fosfato/genética , Simportadores de Próton-Fosfato/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Selenito de Sódio/toxicidadeRESUMO
Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization.
Assuntos
Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/metabolismo , Biologia Computacional/métodos , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Aspartato-tRNA Ligase/genética , Sítios de Ligação , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação Puntual , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genéticaRESUMO
Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Membrana Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Antifúngicos/química , Antifúngicos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Farmacorresistência Fúngica/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato/efeitos dos fármacosRESUMO
GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active d-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous d-Tyr- tRNA(Tyr) deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by d-amino acids are considered.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Acetaldeído/farmacologia , Aminoaciltransferases/genética , Proteínas de Arabidopsis/genética , Catálise , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Etanol/farmacologia , Deleção de Genes , Teste de Complementação Genética , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade por Substrato , Zinco/metabolismoRESUMO
Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand.
Assuntos
Aspartato-tRNA Ligase/metabolismo , Simulação por Computador , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mutagênese Sítio-Dirigida , Aspartato-tRNA Ligase/genética , Sítios de Ligação/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligantes , Ligação Proteica/genética , Eletricidade EstáticaRESUMO
One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication. This article compares these two cases within a single whole-cell model framework. This model relies upon a membrane embedding a chemical reaction network (CRN) synthesizing all the cellular constituents, including the membrane, by feeding from an external nutrient. Assuming the osmolarity is kept constant, the system dynamics are governed by a set of nonlinear differential equations coupling the chemical concentrations and the surface-area-to-volume ratio. The resulting asymptotic trajectories are used to determine the cellular shape by minimizing the membrane bending energy (within an approximate predefined family of shapes). While the stationary case can be handled quite generally, the oscillatory one is investigated using a simple oscillating CRN example, which is used to identify features that are expected to hold for any network. It is found that cellular self-replication can be reached with or without chemical oscillations, and that a requirement common to both stationary and oscillatory cases is that a minimum spontaneous curvature of the membrane is required for the cell to divide once its area and volume are both doubled. The oscillatory case can result in a greater variety of cellular shape trajectories but raises additional constraints for cellular division and self-replication: (i) the ratio of doubling time to oscillation period should be an integer, and (ii) if the oscillation amplitude is sufficiently high, then the spontaneous curvature must be below a maximum value to avoid early division before the end of the cycle. Because of these additional stringent constraints, it is likely that early protocells did not rely upon chemical oscillations. Biochemical oscillations typical of modern evolved cells may have emerged later through evolution for other reasons (e.g., metabolic advantage) and must have required additional feedback mechanisms for such a self-replicating system to be robust against even slight environmental variations (e.g., temperature fluctuations).
Assuntos
Células Artificiais , Divisão Celular , Forma Celular , Modelos BiológicosRESUMO
Selenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress. However, it has become apparent that the mode of action of seleno-compounds varies, depending on its chemical form and speciation. Recent studies in various eukaryotic systems, in particular the model organism Saccharomyces cerevisiae, provide new insights on the cytotoxic mechanisms of selenomethionine and selenocysteine. This review first summarizes current knowledge on reactive oxygen species (ROS)-induced genotoxicity of inorganic selenium species. Then, we discuss recent advances on our understanding of the molecular mechanisms of selenocysteine and selenomethionine cytotoxicity. We present evidences indicating that both oxidative stress and ROS-independent mechanisms contribute to selenoamino acids cytotoxicity. These latter mechanisms include disruption of protein homeostasis by selenocysteine misincorporation in proteins and/or reaction of selenols with protein thiols.
Assuntos
Compostos Organosselênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Células Eucarióticas/efeitos dos fármacos , Redes e Vias Metabólicas , Modelos Biológicos , Selenocisteína/toxicidade , Selenometionina/toxicidadeRESUMO
Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes.
Assuntos
Agregados Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Selenocisteína/metabolismo , Selenometionina/toxicidade , Sequência de Aminoácidos , Bases de Dados como Assunto , Deleção de Genes , Ontologia Genética , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
An enzyme capable of liberating functional tRNA(Lys) from Escherichia coli diacetyl-lysyl-tRNA(Lys) was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA(fMet) as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA(Lys) from diacetyl-lysyl-tRNA(Lys). Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Sulfolobus/enzimologia , Animais , Sequência de Bases , Genes Arqueais , Genoma , Dados de Sequência Molecular , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência , Especificidade por SubstratoRESUMO
Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator. In parallel, the abilities of these mixtures to produce colloids were assessed. Studied metal cations could be classified in three groups: (i) metal ions that protect cells against selenium toxicity and form insoluble colloids with selenide (Agâº, Cd²âº, Cu²âº, Hg²âº, Pb²âº and Zn²âº), (ii) metal ions which protect cells by producing insoluble metal-selenide complexes and by catalyzing hydrogen selenide oxidation in the presence of dioxygen (Co²âº and Ni²âº) and, finally, (iii) metal ions which do not afford protection and do not interact (Ca²âº, Mg²âº, Mn²âº) or weakly interact (Fe²âº) with selenide under the assayed conditions. When occurring, the insoluble complexes formed from divalent metal ions and selenide contained equimolar amounts of metal and selenium atoms. With the monovalent silver ion, the complex contained two silver atoms per selenium atom. Next, because selenides are compounds prone to oxidation, the stabilities of the above colloids were evaluated under oxidizing conditions. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the reduction of which can be optically followed, was used to promote selenide oxidation. Complexes with cadmium, copper, lead, mercury or silver resisted dissolution by DTNB treatment over several hours. With nickel and cobalt, partial oxidation by DTNB occurred. On the other hand, when starting from ZnSe or FeSe complexes, full decompositions were obtained within a few tens of minutes. The above properties possibly explain why ZnSe and FeSe nanoparticles were not detected in animals exposed to selenocompounds.
Assuntos
Coloides/química , Coloides/toxicidade , Metais/química , Metais/toxicidade , Compostos de Selênio/química , Cádmio/química , Cádmio/toxicidade , Cobre/química , Cobre/toxicidade , Intoxicação por Metais Pesados , Níquel/química , Níquel/toxicidade , Intoxicação , Saccharomyces cerevisiae/efeitos dos fármacos , Prata/química , Prata/toxicidadeRESUMO
Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (â¢)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.
Assuntos
Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Oxigênio , Saccharomyces cerevisiae/genética , Compostos de Selênio/toxicidade , Aerobiose , Anaerobiose , Morte Celular/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Técnicas de Inativação de Genes , Genoma Fúngico , Haploidia , Recombinação Homóloga/efeitos dos fármacos , Hipersensibilidade , Manitol/farmacologia , Oxigênio/química , Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Compostos de Selênio/química , Compostos de Selênio/metabolismoRESUMO
Escherichia coli peptidyl-tRNA hydrolase activity is inhibited by 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, a stable mimic of the minimalist substrate 2'(3')-O-(L-[N,N-diacetyl-lysinyl)adenosine. The complex of this mimic with the enzyme has been analyzed by NMR spectroscopy, enabling experimental mapping of the catalytic center for the first time. Chemical shift variations point out the sensitivity of residues Asn10, Met67, Asn68, Gly111, Asn114, Leu116, Lys117, Gly147, Phe148, and Val149 to complex formation. Docking simulations based on ambiguous interaction restraints involving these residues show bondings of the peptide moiety of 3'-(l-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine with Asn10, Asn68, and Asn114. A stacking interaction of Phe66 with the purine is also indicated. Drawn is a model of enzyme-bound peptidyl-tRNA substrate, in which: (i) the Asn114 δ(2) NH(2) group holds the water molecule that participates in the hydrolysis of the substrate, while Tyr15 binds the phosphate in the 5'-position of the 3'-terminal tRNA adenosine; (ii) the δ(2) NH(2) group of Asn68 holds the main-chain carbonyl of the C-terminal residue of the peptide esterified to tRNA; and (iii) the δ(2) NH(2) group of Asn10 holds the main-chain carbonyl of the penultimate C-residue. Functional value is given to this model by (i) showing that the enzyme becomes confusable with an aminoacyl-tRNA hydrolase upon mutagenesis of Asn10 and (ii) reinterpreting already obtained site-directed mutagenesis data.