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1.
J Agric Food Chem ; 67(37): 10521-10533, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461284

RESUMO

This work was designed to comparatively investigate 27 dietary flavonoids that act as α-glucosidase inhibitors and insulin sensitizers. On the basis of the results of an in vitro experiment of α-glucosidase inhibition, myricetin (IC50 = 11.63 ± 0.36 µM) possessed the strongest inhibitory effect, followed by apigenin-7-O-glucoside (IC50 = 22.80 ± 0.24 µM) and fisetin (IC50 = 46.39 ± 0.34 µM). A three-dimensional quantitative structure-activity relationship model of α-glucosidase inhibitors with good predictive capability [comparative molecular field analysis, q2 = 0.529, optimum number of components (ONC) = 10, R2 = 0.996, F = 250.843, standard error of estimation (SEE) = 0.064, and two descriptors; comparative similarity index analysis, q2 = 0.515, ONC = 10, R2 = 0.997, F = 348.301, SEE = 0.054, and four descriptors] was established and indicated that meta positions of ring B favored bulky and minor, electron-withdrawing, and hydrogen bond donor groups. The presence of electron-donating and hydrogen bond acceptor groups at position 4' of ring B could improve α-glucosidase activity. Position 3 of ring C favored minor, electron-donating, and hydrogen bond donor groups, whereas position 7 of ring A favored bulky and hydrogen bond acceptor groups. Molecular docking screened five flavonoids (baicalein, isorhamnetin-3-O-rutinoside, apigenin-7-O-glucoside, kaempferol-7-O-ß-glucoside, and cyanidin-3-O-glucoside) that can act as insulin sensitizers and form strong combinations with four key protein targets involved in the insulin signaling pathway. Apigenin-7-O-glucoside (60 µM) can effectively improve insulin resistance, and glucose uptake increased by approximately 73.06% relative to the model group of insulin-resistant HepG2 cells. Therefore, apigenin-7-O-glucoside might serve as the most effective α-glucosidase inhibitor and insulin sensitizer. This work may guide diabetes patients to improve their condition through dietary therapy.


Assuntos
Flavonoides/química , Inibidores de Glicosídeo Hidrolases/química , Insulina/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacologia , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo
2.
Nat Commun ; 10(1): 2894, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263106

RESUMO

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication.


Assuntos
Nucleossomos/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Montagem e Desmontagem da Cromatina , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo
3.
BMC Bioinformatics ; 20(1): 355, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234779

RESUMO

BACKGROUND: Essential proteins are distinctly important for an organism's survival and development and crucial to disease analysis and drug design as well. Large-scale protein-protein interaction (PPI) data sets exist in Saccharomyces cerevisiae, which provides us with a valuable opportunity to predict identify essential proteins from PPI networks. Many network topology-based computational methods have been designed to detect essential proteins. However, these methods are limited by the completeness of available PPI data. To break out of these restraints, some computational methods have been proposed by integrating PPI networks and multi-source biological data. Despite the progress in the research of multiple data fusion, it is still challenging to improve the prediction accuracy of the computational methods. RESULTS: In this paper, we design a novel iterative model for essential proteins prediction, named Randomly Walking in the Heterogeneous Network (RWHN). In RWHN, a weighted protein-protein interaction network and a domain-domain association network are constructed according to the original PPI network and the known protein-domain association network, firstly. And then, we establish a new heterogeneous matrix by combining the two constructed networks with the protein-domain association network. Based on the heterogeneous matrix, a transition probability matrix is established by normalized operation. Finally, an improved PageRank algorithm is adopted on the heterogeneous network for essential proteins prediction. In order to eliminate the influence of the false negative, information on orthologous proteins and the subcellular localization information of proteins are integrated to initialize the score vector of proteins. In RWHN, the topology, conservative and functional features of essential proteins are all taken into account in the prediction process. The experimental results show that RWHN obviously exceeds in predicting essential proteins ten other competing methods. CONCLUSIONS: We demonstrated that integrating multi-source data into a heterogeneous network can preserve the complex relationship among multiple biological data and improve the prediction accuracy of essential proteins. RWHN, our proposed method, is effective for the prediction of essential proteins.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Algoritmos , Domínios Proteicos , Mapas de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/química
4.
Genes Dev ; 33(15-16): 1031-1047, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196865

RESUMO

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.


Assuntos
Aneuploidia , Citosol/metabolismo , Compensação de Dosagem (Genética)/fisiologia , Agregados Proteicos/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Agregação Patológica de Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nature ; 571(7765): 366-370, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243363

RESUMO

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.


Assuntos
ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Microscopia Crioeletrônica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Transporte Biológico , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/ultraestrutura , Ativação Enzimática , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/ultraestrutura
6.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
7.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 392-396, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045569

RESUMO

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P212121 and its structure solution and refinement to 1.22 Šresolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.


Assuntos
Cisteína/química , Glutarredoxinas/química , Glutationa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
8.
PLoS Genet ; 15(4): e1007786, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946740

RESUMO

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Trissacarídeos/metabolismo , Sequência de Aminoácidos , Cerveja/microbiologia , Proteínas de Transporte/química , Evolução Molecular Direcionada , Fermentação , Proteínas Fúngicas/química , Conversão Gênica , Genes Fúngicos , Hibridização Genética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/química , Saccharomyces/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Simportadores/química , Simportadores/genética , Simportadores/metabolismo
9.
PLoS Genet ; 15(4): e1008079, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30969963

RESUMO

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.


Assuntos
Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aptidão Genética , Leveduras/genética , Leveduras/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Epistasia Genética , Proteínas Fúngicas/química , Genes Fúngicos , Genótipo , Hidroliases/química , Hidroliases/genética , Hidroliases/metabolismo , Modelos Genéticos , Modelos Moleculares , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Comput Biol Chem ; 80: 168-176, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965174

RESUMO

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Quitina Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Antifúngicos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Quitina Sintase/química , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência
11.
Genes Dev ; 33(11-12): 705-717, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30948432

RESUMO

The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.


Assuntos
RNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Mutantes/metabolismo , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
12.
Nucleic Acids Res ; 47(7): 3699-3710, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993346

RESUMO

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.


Assuntos
RNA Helicases DEAD-box/química , RNA/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos/genética , RNA Helicases DEAD-box/genética , Humanos , Fenômenos Mecânicos , RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Nat Commun ; 10(1): 1130, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850607

RESUMO

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1-TRIAP1 and PRELID3b-TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.


Assuntos
Proteínas de Transporte/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Mitocondriais/química , Ácidos Fosfatídicos/química , Fosfatidilserinas/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ácidos Fosfatídicos/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Nat Commun ; 10(1): 1273, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894538

RESUMO

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.


Assuntos
Adenosina Trifosfatases/química , Chaperoninas/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Chaperoninas/genética , Chaperoninas/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
15.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 3): 159-165, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30839289

RESUMO

Osm1, a soluble fumarate reductase from Saccharomyces cerevisiae, is localized in both the mitochondria and the endoplasmic reticulum (ER). OSM1 genetically interacts with ERO1, which encodes an essential ER oxidoreductase for disulfide-bond formation under anaerobic conditions. However, the detailed enzymatic mechanisms involved in this interaction and the cellular roles of Osm1 are not fully understood. In this study, monomeric and stable recombinant Osm1 was successfully prepared for structural study. During purification, it was realized that the majority of recombinant Osm1 expressed in Escherichia coli lacked the flavin adenosine dinucleotide (FAD) cofactor. However, exogenously introduced FAD could be incorporated into recombinant Osm1, generating stable and homogenous holo Osm1. Moreover, after removing a flexible fragment by limited proteolysis, holo Osm1 formed isotropic crystals that retained catalytic activity. X-ray diffraction data were successfully collected from the Osm1 crystals to a resolution of 1.75 Å.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Proteínas de Saccharomyces cerevisiae/química , Succinato Desidrogenase/química , Cristalografia por Raios X , Escherichia coli/genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Succinato Desidrogenase/genética
16.
Chemphyschem ; 20(8): 1011-1017, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30864174

RESUMO

Ligand-mediated regulation of protein assembly occurs frequently in different cellular contexts. Auto-regulated assembly, where a ligand acts as its own competitive inhibitor, provides a mechanism for exquisite control of assembly. Unlike simple protein-ligand systems a quantification of the binding thermodynamics is not straightforward. Here, we characterize the interactions of a recently identified model system in which the oligomerization of cytochrome c is controlled by sulfonato-calix[8]arene, an anionic supramolecular scaffold. Isothermal titration calorimetry and thermodynamic modelling, in combination with Bayesian fitting, were used to quantify the ligand binding and assembly equilibria for this system. The approach and variations of this model may prove useful for the analysis of auto-regulated protein assembly in general.


Assuntos
Calixarenos/química , Citocromos c/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Ânions/química , Teorema de Bayes , Sítios de Ligação , Ligantes , Modelos Moleculares , Multimerização Proteica , Termodinâmica
17.
Genes Genet Syst ; 94(1): 51-59, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30905891

RESUMO

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box. We previously demonstrated that Drosophila TAND1 binds to TBP with a much stronger affinity than yeast TAND1 and that the expression levels of full-length chimeric Taf1p, whose TAND1 is replaced with the Drosophila counterpart, can be varied in vivo by substituting several methionine residues downstream of TAND2 with alanine residues in various combinations. In this study, we examined the transcriptional activation of the GAL1-lacZ reporter or endogenous genes such as RNR3 or GAL1 in yeast cells expressing various levels of full-length chimeric Taf1p. The results showed that the substitution of TAND1 with the Drosophila counterpart in yeast TFIID weakened the transcriptional activation of GAL1-lacZ and RNR3 but not that of GAL1. These findings strongly support a model in which TBP must be released efficiently from TAND1 within TFIID upon transcriptional activation.


Assuntos
Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Ativação Transcricional , Animais , Drosophila melanogaster , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Domínios Proteicos , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética
18.
Mol Cell ; 74(1): 73-87.e8, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30876805

RESUMO

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Elongação Traducional da Cadeia Peptídica , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ressonância Magnética Nuclear Biomolecular , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
19.
Mol Cell ; 74(1): 88-100.e9, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30876804

RESUMO

Eukaryotic elongation factor 2 (eEF2) is an abundant and essential component of the translation machinery. The biogenesis of this 93 kDa multi-domain protein is assisted by the chaperonin TRiC/CCT. Here, we show in yeast cells that the highly conserved protein Hgh1 (FAM203 in humans) is a chaperone that cooperates with TRiC in eEF2 folding. In the absence of Hgh1, a substantial fraction of newly synthesized eEF2 is degraded or aggregates. We solved the crystal structure of Hgh1 and analyzed the interaction of wild-type and mutant Hgh1 with eEF2. These experiments revealed that Hgh1 is an armadillo repeat protein that binds to the dynamic central domain III of eEF2 via a bipartite interface. Hgh1 binding recruits TRiC to the C-terminal eEF2 module and prevents unproductive interactions of domain III, allowing efficient folding of the N-terminal GTPase module. eEF2 folding is completed upon dissociation of TRiC and Hgh1.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
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