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1.
Nat Commun ; 12(1): 2220, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850123

RESUMO

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Complexo Mediador/química , Complexo Mediador/genética , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
2.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652591

RESUMO

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein-protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Repressoras/química , Ressonância de Plasmônio de Superfície , Humanos , Ligação Proteica , Proteínas Repressoras/metabolismo
3.
Nat Commun ; 12(1): 1846, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758180

RESUMO

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.


Assuntos
Evolução Molecular Direcionada/métodos , Genes de Troca , Engenharia Genética/métodos , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genes Reporter , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Razão Sinal-Ruído , Tetraciclina/farmacologia , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , beta Caroteno/biossíntese , beta Caroteno/genética , beta Caroteno/metabolismo
4.
Nat Genet ; 53(4): 511-520, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649594

RESUMO

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.


Assuntos
Fator de Ligação a CCAAT/química , Hemoglobina Fetal/genética , Hemoglobina A/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/química , gama-Globinas/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Eritropoese/genética , Hemoglobina Fetal/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica , Células HEK293 , Hemoglobina A/metabolismo , Humanos , Modelos Moleculares , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células-Tronco , Globinas beta/química , Globinas beta/genética , Globinas beta/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismo
5.
Nature ; 590(7846): 463-467, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536618

RESUMO

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.


Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Furanos/química , Hormônios/química , Hormônios/classificação , Hormônios/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/classificação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relação Estrutura-Atividade
6.
RNA ; 27(4): 465-476, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33408095

RESUMO

The 3' to 5' exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but Saccharomyces cerevisiae Pop2p harbors noncanonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S. cerevisiae Pop2p. We find that S. cerevisiae Pop2p degrades RNAs via two alternative reactions pathways, one generating nucleotides with 5'-phosphates and RNA intermediates with 3'-hydroxyls, and the other generating nucleotides with 3'-phosphates and RNA intermediates with 3'-phosphates. The enzyme is not able to initiate the reaction on RNAs with a 3'-phosphate, which leads to accumulation of RNAs with 3'-phosphates that can exceed 10 nt and are resistant to further degradation by S. cerevisiae Pop2p. We further demonstrate that S. cerevisiae Pop2p degrades RNAs in three reaction phases: an initial distributive phase, a second processive phase and a third phase during which processivity gradually declines. We also show that mutations of subsets of amino acids in the catalytic center, including those previously thought to inactivate the enzyme, moderately reduce, but not eliminate activity. Only mutation of all five amino acids in the catalytic center diminishes activity of Pop2p to background levels. Collectively, our results reveal robust exonuclease activity of S. cerevisiae Pop2p with unusual enzymatic properties, characterized by alternative degradation pathways, multiple reaction phases and functional redundancy of amino acids in the catalytic core.


Assuntos
Aminoácidos/química , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Monofosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutação , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
7.
J Mol Biol ; 433(4): 166806, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33450246

RESUMO

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Reparo do DNA , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Linhagem Celular , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
J Mol Biol ; 433(4): 166812, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33450249

RESUMO

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/química , Dineínas/química , Cinetocoros/química , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância Magnética , Ligação Proteica , Conformação Proteica , Fuso Acromático/metabolismo , Relação Estrutura-Atividade
9.
Mutat Res ; 785: 108319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32800270

RESUMO

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fenda Labial/genética , Fissura Palatina/genética , Proteínas Repressoras/genética , Fumar/efeitos adversos , Motivos de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Metilação de DNA , Suplementos Nutricionais , Feminino , Ácido Fólico/metabolismo , Estudos de Associação Genética , Humanos , Recém-Nascido , Masculino , Modelos Moleculares , Domínios Proteicos , Isoformas de RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fatores de Risco
10.
Infect Immun ; 88(11)2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32817331

RESUMO

Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.


Assuntos
Proteínas de Bactérias/genética , Fasciite Necrosante/microbiologia , Histidina Quinase/genética , Miosite/microbiologia , Proteínas Repressoras/genética , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Transativadores/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fasciite Necrosante/metabolismo , Fasciite Necrosante/mortalidade , Fasciite Necrosante/patologia , Feminino , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Humanos , Camundongos , Mutação , Miosite/metabolismo , Miosite/mortalidade , Miosite/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/mortalidade , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Análise de Sobrevida , Transativadores/química , Transativadores/metabolismo , Virulência
11.
Proc Natl Acad Sci U S A ; 117(34): 20576-20585, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788352

RESUMO

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R 1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.


Assuntos
Bacteriófagos/fisiologia , Lisogenia , Proteínas Repressoras/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Genoma Bacteriano , Interações Hospedeiro-Patógeno , Cinética , Lactococcus lactis/virologia , Simulação de Dinâmica Molecular , Regiões Operadoras Genéticas , Conformação Proteica , Proteínas Repressoras/química , Proteínas Virais Reguladoras e Acessórias/química
12.
Nucleic Acids Res ; 48(17): 9571-9588, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32813023

RESUMO

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Repressoras/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Linhagem Celular , DNA Bacteriano/metabolismo , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Peptidilprolil Isomerase/genética , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/metabolismo
13.
Nat Struct Mol Biol ; 27(9): 836-845, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747783

RESUMO

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Nucleossomos/metabolismo , Nucleossomos/patologia , Proteínas de Fusão Oncogênica/química , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Sarcoma Sinovial/patologia , Fatores de Transcrição/química , Ubiquitinação
14.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , 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 , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
15.
Nature ; 585(7824): 256-260, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848244

RESUMO

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Priônicas/química , Temperatura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Aclimatação/fisiologia , Arabidopsis/química , Temperatura Alta , Modelos Moleculares , Peptídeos/metabolismo , Transição de Fase , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo
16.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
17.
Proc Natl Acad Sci U S A ; 117(20): 10989-10999, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32354997

RESUMO

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas , Sequência de Bases , Biofilmes , Domínio Catalítico , Modelos Animais de Doenças , Endocardite , Enterotoxinas , Feminino , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/metabolismo , Masculino , Modelos Moleculares , Mutação , Oxirredução , Domínios Proteicos , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sepse , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Superantígenos , Thermotoga maritima , Virulência/genética , Virulência/fisiologia
18.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
19.
Appl Environ Microbiol ; 86(11)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32245756

RESUMO

Acidophiles play a dominant role in driving elemental cycling in natural acid mine drainage (AMD) habitats and exhibit important application value in bioleaching and bioremediation. Acidity is an inevitable environmental stress and a key factor that affects the survival of acidophiles in their acidified natural habitats; however, the regulatory strategies applied by acidophilic bacteria to withstand low pH are unclear. We identified the significance of the ferric uptake regulator (Fur) in acidophiles adapting to acidic environments and discovered that Fur is ubiquitous as well as highly conserved in acidophilic bacteria. Mutagenesis of the fur gene of Acidithiobacillus caldus, a prototypical acidophilic sulfur-oxidizing bacterium found in AMD, revealed that Fur is required for the acid resistance of this acidophilic bacterium. Phenotypic characterization, transcriptome sequencing (RNA-seq), mutagenesis, and biochemical assays indicated that the Acidithiobacillus caldus ferric uptake regulator (AcFur) is involved in extreme acid resistance by regulating the expression of several key genes of certain cellular activities, such as iron transport, biofilm formation, sulfur metabolism, chemotaxis, and flagellar biosynthesis. Finally, a Fur-dependent acid resistance regulatory strategy in A. caldus was proposed to illustrate the ecological behavior of acidophilic bacteria under low pH. This study provides new insights into the adaptation strategies of acidophiles to AMD ecosystems and will promote the design and development of engineered biological systems for the environmental adaptation of acidophiles.IMPORTANCE This study advances our understanding of the acid tolerance mechanism of A. caldus, identifies the key fur gene responsible for acid resistance, and elucidates the correlation between fur and acid resistance, thus contributing to an understanding of the ecological behavior of acidophilic bacteria. These findings provide new insights into the acid resistance process in Acidithiobacillus species, thereby promoting the study of the environmental adaptation of acidophilic bacteria and the design of engineered biological systems.


Assuntos
Acidithiobacillus/fisiologia , Adaptação Biológica/genética , Proteínas de Bactérias/genética , Ecossistema , Concentração de Íons de Hidrogênio , Proteínas Repressoras/genética , Acidithiobacillus/genética , Ácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Compostos Férricos/metabolismo , Mineração , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Alinhamento de Sequência
20.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32332134

RESUMO

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization.IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.


Assuntos
Proteínas de Bactérias/genética , Endófitos/genética , Enterobacteriaceae/genética , Proteínas Repressoras/genética , Transativadores/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Endófitos/metabolismo , Enterobacteriaceae/metabolismo , Oryza/microbiologia , Filogenia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Simbiose/genética , Transativadores/química , Transativadores/metabolismo
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