RESUMO
Metschnikowia reukaufii is a widespread yeast able to grow in the plants' floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l-1 , which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1â1), α-(1â3) and α-(1â6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l-1 , the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l-1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.
Assuntos
Glucosiltransferases/metabolismo , Metschnikowia/enzimologia , Metschnikowia/metabolismo , Trissacarídeos/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Trissacarídeos/química , Trissacarídeos/isolamento & purificaçãoRESUMO
BACKGROUND: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. RESULTS: Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5-6.5 and 30-40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (D-glucosamine)1-8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. CONCLUSIONS: Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.
Assuntos
Quitina/análogos & derivados , Quitina/química , Quitinases/química , Quitosana/química , Proteínas/química , OligossacarídeosRESUMO
The ribosomal sarcin/ricin loop (SRL) is the target of ribosome-inactivating proteins like the N-glycosidase ricin and the fungal ribotoxin α-sarcin. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL. Here we tested this hypothesis for the ribotoxin α-sarcin. Experiments with isolated ribosomes, cell-free translation systems, and viability assays with Saccharomyces cerevisiae strains defective in acidic stalk proteins showed that the inactivation exerted by α-sarcin is independent of the composition of the ribosomal stalk. Therefore, α-sarcin, with the same ribosomal target as ricin, seems to access the SRL by a different pathway.
Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/genética , Ricina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The P0 scaffold protein of the ribosomal stalk is mainly incorporated into pre-ribosomes in the cytoplasm where it replaces the assembly factor Mrt4. In analyzing the role of the P0 carboxyl terminal domain (CTD) during ribosomal stalk assembly, we found that its complete removal yields a protein that is functionally similar to Mrt4, whereas a chimeric Mrt4 containing the P0 CTD behaves more like P0. Deleting the P0 binding sites for the P1 and P2 proteins provoked the nuclear accumulation of P0ΔAB induced by either leptomycin B-mediated blockage of nuclear export or Mrt4 deletion. This effect was reversed by removing P1/P2 from the cell, whereas nuclear accumulation was restored on reintroduction of these proteins. Together, these results indicate that the CTD determines the function of the P0 in stalk assembly. Moreover, they indicate that in cells lacking Mrt4, P0 and its stalk base partner, the L12 protein, bind to pre-ribosomes in the nucleus, a complex that is then exported to the cytoplasm by a mechanism assisted by the interaction with P1/P2 proteins. Furthermore, in wild-type cells, the presence of nuclear pre-ribosome complexes containing P0 but not L12 is compatible with the existence of an alternative stalk assembly process.
Assuntos
Núcleo Celular/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Corantes Fluorescentes , Deleção de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Fracionamento Celular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade da EspécieRESUMO
The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1ß, P2α and P2ß, which form two heterodimers, P1α/P2ß and P1ß/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1ß-P2ß heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1ß/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2ß-P0-P1ß/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.
Assuntos
Subunidades Proteicas/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Dimerização , Mutação , Fenótipo , Polirribossomos/metabolismo , Subunidades Proteicas/genética , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2ßin vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.
Assuntos
Purinas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Sítios de Ligação , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutação , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae/genética , Toxina Shiga I/farmacologia , Toxina Shiga II/farmacologiaRESUMO
The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2ß four initial amino acids, while these mutations have no effect on P1ß. Binding of P2ß and, to a lesser extent, P1ß to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation.
Assuntos
Fosfoproteínas/química , Proteínas Ribossômicas/química , Proteínas de Saccharomyces cerevisiae/química , Dimerização , Modelos Moleculares , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Heterodimers of acidic proteins P1alpha/P2beta and P1beta/P2alpha bind to P0 and are fundamental for the assembly of the ribosomal stalk. However, different inconsistencies are found in the literature regarding additional P protein heterodimer formations and their individual interactions with P0. Using the two-hybrid approach, we have found results that help to clarify these interactions. Thus, we have found that neither P1 nor P2 directly interact with P0 unless the endogenous heterodimer partner is being expressed in the cell. In addition, a P2-free amino end is a requisite in these heterodimers for binding to P0. With regard to the two-hybrid interactions between P1 and P2, the known canonical P1alpha-P2beta and P1beta-P2alpha interactions do not depend on either a free amino end or the presence of endogenous P0, P1 or P2 proteins. Furthermore, the non-canonical P1beta-P2beta pair also behaves similarly, although this interaction is weaker. Interestingly, P1alpha-P2alpha, P1alpha-P1beta and P2alpha-P2beta two-hybrid interactions were also detected, although in these cases the endogenous P proteins were involved. Thus, these positive interactions are the consequence of the interaction between two canonical heterodimers. As the ribosome anchorage protein P0 is also necessary, the results suggest that, in vivo, all five P proteins form a complex, independent of the ribosome, containing the two canonical heterodimers and P0. This complex has been isolated in cells expressing a P0 protein unable to bind to the ribosome.
Assuntos
Fosfoproteínas/metabolismo , Multimerização Proteica , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mrt4 is a nucleolar component of the ribosome assembly machinery that shares notable similarity and competes for binding to the 25S rRNA GAR domain with the ribosomal protein P0. Here, we show that loss of function of either P0 or Mrt4 results in a deficit in 60S subunits, which is apparently due to impaired rRNA processing of 27S precursors. Mrt4, which shuttles between the nucleus and the cytoplasm, defines medium pre-60S particles. In contrast, P0 is absent from medium but present in late/cytoplasmic pre-60S complexes. The absence of Mrt4 notably increased the amount of P0 in nuclear Nop7-TAP complexes and causes P0 assembly to medium pre-60S particles. Upon P0 depletion, Mrt4 is relocated to the cytoplasm within aberrant 60S subunits. We conclude that Mrt4 controls the position and timing of P0 assembly. In turn, P0 is required for the release of Mrt4 and exchanges with this factor at the cytoplasm. Our results also suggest other P0 assembly alternatives.
Assuntos
Proteínas Ribossômicas/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Deleção de Genes , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.
Assuntos
Proteínas Ribossômicas/química , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , Modelos Moleculares , Estrutura Terciária de Proteína , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the alpha-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild-type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild-type ribosomes did not follow a single-step binding model but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable, and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable, and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild-type ribosomes. Both interactions were dominated by electrostatic interactions, and the AB1 interaction was stronger than the AB2 interaction. On the basis of these results, we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA toward the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.
Assuntos
Modelos Químicos , Ribossomos/metabolismo , Ricina/metabolismo , Ligantes , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribossomos/química , Ricina/química , Eletricidade Estática , Ressonância de Plasmônio de SuperfícieRESUMO
The essential ribosomal stalk is formed in eukaryotes by a pentamer of two P1-P2 protein heterodimers and the P0 rRNA binding protein. In contrast to the highly stable prokaryotic complex, the P1 and P2 proteins in the eukaryotic stalk undergo a cyclic process of assembly and disassembly during translation that seems to modulate the ribosome activity. To better understand this process, the regions of the Saccharomyces cerevisiae P1alpha and P2beta proteins that are directly involved in heterodimer formation and ribosome binding have been characterized using a series of P1alpha/P2beta chimeras. The region required for a stable interaction with the ribosome is formed by the first three predicted alpha-helices in the N-terminal domain of both proteins. The same region is required for heterodimer formation in P2beta but the third helix is dispensable for this association in P1alpha. It seems, therefore, that stable ribosome binding is more structurally demanding than heterodimerization. A fourth predicted alpha-helix in the N-terminal domain of P1alpha and P2beta appears not to be involved in the assembly process but rather, it contributes to the conformation of the proteins by apparently restricting the mobility of their C-terminal domain and paradoxically, by reducing their activity. In addition, the study of P1/P2 chimeras showed that the C-terminal domains of these two types of protein are functionally identical and that their protein specificity is exclusively determined by their N-terminal domains.
Assuntos
Fosfoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/genética , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
Assuntos
Fosfoproteínas/efeitos dos fármacos , RNA Ribossômico/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ricina/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Genes , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Purinas/química , Purinas/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
A human cell line, in which expression of the ribosomal stalk proteins P1 and P2 has been suppressed by RNAi technology, has been used to test how the loss of these proteins affects IRES-dependent translation. Foot-and-mouth disease virus (FMDV) IRES-dependent translation from a bicistronic construct is about three fold higher in the P1/P2-depleted cells than in control cells in the presence of Lb protease. By contrast, no effect on Hepatitis C virus (HCV) IRES translation was observed. These results emphasize the functional heterogeneity of the IRES and they highlight a functional connection between the ribosomal stalk and picornavirus IRES-dependent translation.
Assuntos
Vírus da Febre Aftosa/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fosfoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/virologia , Sítios de Ligação , Linhagem Celular , Doxiciclina/farmacologia , Vírus da Febre Aftosa/genética , Humanos , Fosfoproteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/metabolismoRESUMO
The 'stalk' is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.
Assuntos
Fosfoproteínas/fisiologia , Proteínas Ribossômicas/fisiologia , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Sordarin and its derivatives are antifungal compounds of potential clinical interest. Despite the highly conserved nature of the fungal and mammalian protein synthesis machineries, sordarin is a selective inhibitor of protein synthesis in fungal organisms. In cells sensitive to sordarin, its mode of action is through preventing the release of translation elongation factor 2 (eEF2) during the translocation step, thus blocking protein synthesis. To further investigate the cellular components required for the effects of sordarin in fungal cells, we have used the haploid deletion collection of Saccharomyces cerevisiae to systematically identify genes whose deletion confers sensitivity or resistance to the compound. Our results indicate that genes in a number of cellular pathways previously unknown to play a role in sordarin response are involved in its growth effects on fungal cells and reveal a specific requirement for the diphthamidation pathway of cells in causing eEF2 to be sensitive to the effects of sordarin on protein synthesis. Our results underscore the importance of the powerful genomic tools developed in yeast (Saccharomyces cerevisiae) to more comprehensively understanding the cellular mechanisms involved in the response to therapeutic agents.
Assuntos
Indenos/farmacologia , Fator 2 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Mutação , Fator 2 de Elongação de Peptídeos/genética , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1alpha, P1beta, P2alpha, and P2beta. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Microscopia de Fluorescência/métodos , Ribossomos/química , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic ribosomal stalk protein L12 and its bacterial orthologue L11 play a central role on ribosomal conformational changes during translocation. Deletion of the two genes encoding L12 in Saccharomyces cerevisiae resulted in a very slow-growth phenotype. Gene RPL12B, but not the RPL12A, cloned in centromeric plasmids fully restored control protein level and the growth rate when expressed in a L12-deprived strain. The same strain has been transformed to express Escherichia coli protein EcL11 under the control of yeast RPL12B promoter. The bacterial protein has been found in similar amounts in washed ribosomes from the transformed yeast strain and from control E. coli cells, however, EcL11 was unable to restore the defective acidic protein stalk composition caused by the absence of ScL12 in the yeast ribosome. Protein EcL11 induced a 10% increase in L12-defective cell growth rate, although the in vitro polymerizing capacity of the EcL11-containing ribosomes is restored in a higher proportion, and, moreover, the particles became partially sensitive to the prokaryotic specific antibiotic thiostrepton. Molecular dynamic simulations using modelled complexes support the correct assembly of bacterial L11 into the yeast ribosome and confirm its direct implication of its CTD in the binding of thiostrepton to ribosomes.
Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/química , Proteínas Ribossômicas/química , Ribossomos/química , Saccharomyces cerevisiae/genética , Tioestreptona/farmacologia , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , RNA Ribossômico/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The stalk is an essential domain of the large ribosomal subunit formed by a complex of a set of very acidic proteins bound to a core rRNA binding component. While in prokaryotes there is only one type acidic protein, L7/12, two protein families are found in eukaryotes, phosphoproteins P1 and P2, which presumably have different roles. To search for differences zero-length cross-linking by S-S bridge formation was applied using Saccharomyces cerevisiae mutant P1 and P2 proteins carrying single cysteine residues at various positions. The results show a more exposed location of the N-terminal domain of the P2 proteins, which in contrast to P1, can be found as dimers when the Cys is introduced in this domain. Similarly, the Cys containing C-terminal domain of mutant P2 proteins shows a notable capacity to form cross-links with other proteins, which is considerably lower in the P1 type. On the other hand, mutation at the conserved C-domain of protein P0, the eukaryotic stalk rRNA binding component, results in removal of about 14 terminal amino acids. Protein P2, but not P1, protects mutant P0 from this truncation. These results support a eukaryotic stalk structure in which P1 proteins are internally located with their C-terminals having a restricted reactivity while P2 proteins are more external and accessible to interact with other cellular components.