Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.988
Filtrar
1.
PLoS One ; 15(7): e0235033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639961

RESUMO

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.


Assuntos
Adenosina Trifosfatases/genética , Antimaníacos/farmacologia , Cloreto de Lítio/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fosfoglucomutase/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
2.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
3.
Nat Commun ; 11(1): 3474, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651392

RESUMO

RNase MRP is an essential eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle. RNase MRP is related to the ribozyme-based RNase P, but it has evolved to have distinct cellular roles. We report a cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme solved to 3.0 Å. We describe the structure of this 450 kDa complex, interactions between its components, and the organization of its catalytic RNA. We show that some of the RNase MRP proteins shared with RNase P undergo an unexpected RNA-driven remodeling that allows them to bind to divergent RNAs. Further, we reveal how this RNA-driven protein remodeling, acting together with the introduction of new auxiliary elements, results in the functional diversification of RNase MRP and its progenitor, RNase P, and demonstrate structural underpinnings of the acquisition of new functions by catalytic RNPs.


Assuntos
Microscopia Crioeletrônica , Endorribonucleases/ultraestrutura , Ribonucleoproteínas/ultraestrutura , Carbono/química , Catálise , Domínio Catalítico , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Catalítico/química , RNA Fúngico/química , Ribonuclease P/química , Saccharomyces cerevisiae/enzimologia
4.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
5.
Proc Natl Acad Sci U S A ; 117(31): 18459-18469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694211

RESUMO

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
7.
PLoS Genet ; 16(6): e1008840, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579556

RESUMO

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1's essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell's energy resources are converted into structural carbohydrates in response to changing cellular needs.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Metabolismo Energético , Glucose/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Mutação , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas de Saccharomyces cerevisiae/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo
8.
Nat Commun ; 11(1): 3156, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572031

RESUMO

The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Šcryo-EM structure of yeast Pol ε revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.


Assuntos
DNA Polimerase II/química , Replicação do DNA , Microscopia Crioeletrônica , Modelos Moleculares , Estrutura Molecular , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Science ; 369(6504): 656-663, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32586950

RESUMO

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.


Assuntos
Endorribonucleases/química , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Ribonucleases/química , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Holoenzimas/química , Conformação Proteica , RNA Catalítico/química , Especificidade por Substrato
10.
PLoS One ; 15(5): e0232755, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401766

RESUMO

The quality control of intracellular proteins is achieved by degrading misfolded proteins which cannot be refolded by molecular chaperones. In eukaryotes, such degradation is handled primarily by the ubiquitin-proteasome system. However, it remained unclear whether and how protein quality control deploys various deubiquitinases. To address this question, we screened deletions or mutation of the 20 deubiquitinase genes in Saccharomyces cerevisiae and discovered that almost half of the mutations slowed the removal of misfolded proteins whereas none of the remaining mutations accelerated this process significantly. Further characterization revealed that Ubp6 maintains the level of free ubiquitin to promote the elimination of misfolded cytosolic proteins, while Ubp3 supports the degradation of misfolded cytosolic and ER luminal proteins by different mechanisms.


Assuntos
Citosol/enzimologia , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Aneuploidia , Degradação Associada com o Retículo Endoplasmático , Testes Genéticos , Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo
11.
Int J Food Microbiol ; 326: 108648, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32387971

RESUMO

Consumption of fructan-containing cereal products is considered beneficial for most people, but not for those suffering from irritable bowel syndrome (IBS), as they should avoid the consumption of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (acronym: FODMAP). Controlling fructan levels in cereal products is not trivial. However, controlling yeast invertase-mediated hydrolysis of fructan during dough fermentation might offer a handle to modulate fructan concentrations. In this work, the variability in invertase activity and substrate specificity in an extensive set of industrial Saccharomyces cerevisiae strains is investigated. Analysis showed a high variability in the capacity of these strains to hydrolyse sucrose and fructo-oligosaccharides. Industrial yeast strains with a high activity towards fructo-oligosaccharides efficiently reduced wheat grain fructans during dough fermentation to a final fructan level of 0.3% dm, whereas strains with a low invertase activity yielded fructan levels around 0.6% dm. The non-bakery strains produced lower levels of CO2 in fermenting dough resulting in lower loaf volumes. However, CO2 production and loaf volume could be increased by the addition of 3% glucose. In conclusion, this study shows that variation in yeast invertase activity and specificity can be used to modulate the fructan content in bread, allowing the production of low FODMAP breads, or alternatively, breads with a higher soluble dietary fibre content.


Assuntos
Dissacarídeos/análise , Monossacarídeos/análise , Oligossacarídeos/análise , Saccharomyces cerevisiae/enzimologia , Triticum/química , beta-Frutofuranosidase/metabolismo , Pão/análise , Fermentação , Frutanos/análise , Frutanos/metabolismo , Hidrólise , Síndrome do Intestino Irritável/patologia , Sacarose/metabolismo , Fermento Seco
12.
Nucleic Acids Res ; 48(11): 6353-6366, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396195

RESUMO

Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/química , Endopeptidases/química , Endopeptidases/metabolismo , Endorribonucleases/química , Holoenzimas/química , Holoenzimas/metabolismo , Ligases/metabolismo , Modelos Moleculares , Organelas/enzimologia , Organelas/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
13.
Nucleic Acids Res ; 48(11): 6092-6107, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402080

RESUMO

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1's role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Securina/metabolismo , Separase/metabolismo , Transdução de Sinais , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/deficiência , Segregação de Cromossomos , Reparo do DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fase G2 , Deleção de Genes , Mitose , Proteínas Serina-Treonina Quinases/deficiência , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Transcrição Genética , Complexos Ubiquitina-Proteína Ligase/metabolismo
14.
Nat Commun ; 11(1): 1725, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265442

RESUMO

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.


Assuntos
Glutarredoxinas/química , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos/genética , Catálise , Domínio Catalítico/genética , Dissulfetos/química , Ativação Enzimática , Ensaios Enzimáticos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/química , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica em alfa-Hélice , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
PLoS Genet ; 16(4): e1008330, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324744

RESUMO

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.


Assuntos
Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alelos , Anticódon , Citosol/enzimologia , Teste de Complementação Genética , Humanos , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Alinhamento de Sequência , Especificidade por Substrato
16.
PLoS Genet ; 16(4): e1008600, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32343701

RESUMO

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.


Assuntos
Calcineurina/metabolismo , Ciclo Celular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação para Baixo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
Phys Chem Chem Phys ; 22(12): 6749-6754, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32167106

RESUMO

The enzymatic activity of alcohol dehydrogenase (ADH) in the presence of a range of electrolytes is investigated. In the presence of 150 and 200 mM cations a substantial increase in activity following the series GnCl < CsCl < KCl ∼ NaCl < LiCl was observed with a 69% increase in the presence of KCl 200 mM with respect to the salt-free solution. In the presence of 150 and 200 mM anions the increase in activity followed an ion specific trend NaF ∼ NaCl ∼ NaBr > no salt > NaClO4 > NaSCN with a peak in activity increase of 75% in the presence of NaBr. The values of the Michaelis-Menten constant (Km) did not show any significant ion specific effect, while the maximum rate (Vmax) of ethanol oxidation to acetaldehyde was strongly ion specific. The changes in specific activity and Vmax in the presence of anions likely arises from ion specific interactions with charged residues in the active site of ADH. The data indicate that the enzymatic activity of alcohol dehydrogenase can be modulated by the nature of electrolytes at physiological concentration.


Assuntos
Álcool Desidrogenase/metabolismo , Eletrólitos/química , Saccharomyces cerevisiae/enzimologia , Álcool Desidrogenase/química , Eletrólitos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Etanol/metabolismo , Oxirredução
18.
J Med Chem ; 63(7): 3538-3551, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32134266

RESUMO

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.


Assuntos
Inibidores Enzimáticos/química , Glicogênio Sintase/antagonistas & inibidores , Imidazóis/química , Pirazóis/química , Animais , Caenorhabditis elegans/enzimologia , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Cinética , Estrutura Molecular , Ligação Proteica , Pirazóis/síntese química , Pirazóis/metabolismo , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
19.
J Food Sci ; 85(4): 1353-1360, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32220140

RESUMO

Here, molecular docking simulation was used to predict and compare interactions between a recombinant Trametes sp. C30 laccase from Saccharomyces cerevisiae and four aflatoxins (AFB1 , AFB2 , AFG1 , and AFG2 ) as well as their degradation at a molecular level. The computational result of docking simulation indicates that each of the aflatoxins tested can interact with laccase with a binding ability of AFB1 >AFG2 >AFG1 >AFB2 . Simultaneously, it also demonstrated that aflatoxin B1 , B2 , G1 , G2 may interact near the T1 copper center of the enzyme through H-bonds and hydrophobic interactions with amino acid residues His481 and Asn288; His481; Asn288, and Asp230; His481 and Asn288. Biological degradation test was performed in vitro in the presence of a recombinant laccase. Degradation increased as incubation time increased from 12 to 60 hr and the maximum degradation obtained for AFB1 , AFB2 , AFG1 , and AFG2 was 90.33%, 74.23%, 85.24%, and 87.58%, respectively. Maximum degradation of aflatoxins was determined with a total activity 3 U laccase at 30 °C in 0.1 M phosphate buffer, pH 5.7 after 48-hr incubation. The experimental results are consistent with that of docking calculation on the biological degradation test of four aflatoxins by laccase. PRACTICAL APPLICATION: In this study, the degradation efficiencies of laccase for B and G series of aflatoxins were determined by computer simulation and verified by performing in vitro experiments. It can provide reference for rapid screening of aflatoxin degradation-related enzymes.


Assuntos
Aflatoxinas/metabolismo , Contaminação de Alimentos/análise , Lacase/metabolismo , Saccharomyces cerevisiae/enzimologia , Trametes/enzimologia , Aflatoxina B1/química , Aflatoxinas/análise , Aflatoxinas/química , Cromatografia Líquida de Alta Pressão/métodos , Simulação por Computador , Lacase/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular
20.
Nat Commun ; 11(1): 688, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019936

RESUMO

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.


Assuntos
Replicação do DNA , Saccharomyces cerevisiae/enzimologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 4 do Complexo de Manutenção de Minicromossomo/química , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA