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1.
Chem Biol Interact ; 311: 108796, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421116

RESUMO

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5'-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1-3 and 5'-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin. Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.


Assuntos
Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nitrilos/farmacologia , Nucleotidases/genética , Nucleosídeos de Purina/metabolismo , Piretrinas/farmacologia , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Masculino , Espectrometria de Massas , Nitrilos/sangue , Nitrilos/metabolismo , Nucleotidases/metabolismo , Piretrinas/sangue , Piretrinas/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Ratos Wistar
2.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
3.
BMC Plant Biol ; 19(1): 282, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248374

RESUMO

BACKGROUND: Heavy metal toxicity has become a major threat to sustainable crop production worldwide. Thus, considerable interest has been placed on deciphering the mechanisms that allow plants to combat heavy metal stress. Strategies to deal with heavy metals are largely focused on detoxification, transport and/or sequestration. The P1B subfamily of the Heavy Metal-transporting P-type ATPases (HMAs) was shown to play a crucial role in the uptake and translocation of heavy metals in plants. Here, we report the locus-specific expression changes in the rice HMA genes together with several low-copy cellular genes and transposable elements upon the heavy metal treatment and monitored the transgenerational inheritance of the altered expression states. We reveal that plants cope with heavy metal stress by making heritable changes in gene expression and further determined gene-specific responses to heavy metal stress. RESULTS: We found most HMA genes were upregulated in response to heavy metal stress, and furthermore found evidence of transgenerational memory via changes in gene regulation even after the removal of heavy metals. To explore whether DNA methylation was also altered in response to the heavy metal stress, we selected a Tos17 retrotransposon for bisulfite sequencing and studied its methylation state across three generations. We found the DNA methylation state of Tos17 was altered in response to the heavy metal stress and showed transgenerational inheritance. CONCLUSIONS: Collectively, the present study elucidates heritable changes in gene expression and DNA methylation in rice upon exposure to heavy metal stress and discusses implications of this knowledge in breeding for heavy metal tolerant crops.


Assuntos
Adenosina Trifosfatases/genética , Epigênese Genética/genética , Expressão Gênica/genética , Metais Pesados/efeitos adversos , Oryza/genética , Proteínas de Plantas/genética , Poluentes do Solo/efeitos adversos , Adenosina Trifosfatases/metabolismo , Oryza/enzimologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico
4.
Nat Commun ; 10(1): 2872, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253804

RESUMO

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short ß-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Cristalização , Escherichia coli , Geobacillus/metabolismo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC/genética
5.
Mol Med Rep ; 19(6): 5162-5168, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059026

RESUMO

Doxorubicin is one of the most widely used chemotherapy agents for the treatment of breast cancer. However, the development of doxorubicin resistance limits the long­term treatment benefits in patients with breast cancer. Curcumin, a well­known dietary polyphenol derived from the rhizomes of turmeric (Curcuma longa), enhances the sensitivity of breast cancer cells to chemotherapeutic agents; however, the mechanisms underlying this phenomenon remain unclear. The aim of the present study was to evaluate the effect of curcumin on chemoresistance in doxorubicin­resistant breast cancerMCF­7/DOX and MDA­MB­231/DOX cell lines. Cell Counting Kit­8, monolayer transport, western blot and ATPase activity assays were performed during the study. The results revealed that curcumin significantly enhanced the effect of doxorubicin in doxorubicin­resistant breast cancer cells. The intracellular accumulation of doxorubicin was substantially increased following curcumin treatment in doxorubicin­resistant breast cancer cells, in a manner that was inversely dependent on the activity of ATP binding cassette subfamily B member 4 (ABCB4). Treatment with a combination of curcumin and doxorubicin decreases the efflux of doxorubicin in ABCB4­overexpressing cells. Furthermore, curcumin inhibited the ATPase activity of ABCB4 without altering its protein expression. In conclusion, curcumin reversed doxorubicin resistance in human breast cancer MCF­7/DOX and MDA­MB­231/DOX cells by inhibiting the ATPase activity of ABCB4. The study highlights the promising use of curcumin as a chemosensitizer in the treatment of breast cancer.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Curcumina/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Cães , Feminino , Humanos , Células MCF-7 , Células Madin Darby de Rim Canino
6.
Gene ; 706: 172-180, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31082499

RESUMO

Molecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2∆, dss1∆, and afg3∆) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.


Assuntos
Longevidade/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Envelhecimento , DNA Mitocondrial/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Genes Mitocondriais/genética , Genótipo , Proteínas Mitocondriais/genética , Estresse Oxidativo , Fenótipo , Bombas de Próton/genética , Bombas de Próton/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Transdução de Sinais
7.
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
8.
Nature ; 569(7754): 136-140, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30996347

RESUMO

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica
9.
Nat Genet ; 51(5): 885-895, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962619

RESUMO

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.


Assuntos
Triticum/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Cromossomos de Plantas/genética , Domesticação , Variação Genética , Genoma de Planta , Filogenia , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Seleção Genética , Sintenia , Tetraploidia , Triticum/classificação , Triticum/metabolismo
11.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965578

RESUMO

The identification of gene(s) that are involved in Cd accumulation/tolerance is vital in developing crop cultivars with low Cd accumulation. We developed a doubled haploid (DH) population that was derived from a cross of Suyinmai 2 (Cd-sensitive) × Weisuobuzhi (Cd-tolerant) to conduct quantitative trait loci (QTL) mapping studies. We assessed chlorophyll content, traits that are associated with development, metal concentration, and antioxidative enzyme activity in DH population lines and parents under control and Cd stress conditions. A single QTL, designated as qShCd7H, was identified on chromosome 7H that was linked to shoot Cd concentration; qShCd7H explained 17% of the phenotypic variation. Comparative genomics, map-based cloning, and gene silencing were used in isolation, cloning, and functional characterization of the candidate gene. A novel gene HvPAA1, being related to shoot Cd concentration, was identified from qShCd7H. Sequence comparison indicated that HvPAA1 carried seven domains with an N-glycosylation motif. HvPAA1 is predominantly expressed in shoots. Subcellular localization verified that HvPAA1 is located in plasma membrane. The silencing of HvPAA1 resulted in growth inhibition, greater Cd accumulation, and a significant decrease in Cd tolerance. We conclude HvPAA1 is a novel plasma membrane-localized ATPase that contributes to Cd tolerance and accumulation in barley. The results provide us with new insights that may aid in the screening and development of Cd-tolerant and low-Cd-accumulation crops.


Assuntos
Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Hordeum/metabolismo , Adenosina Trifosfatases/genética , Cromossomos de Plantas/genética , Ligação Genética , Haploidia , Fenótipo , Locos de Características Quantitativas/genética
12.
Environ Toxicol Pharmacol ; 69: 72-79, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30965278

RESUMO

Nanoparticle (NP) forms of aluminium oxide (Al2O3) are used in various fields such as engineering, pharmacy, medicine etc. Compounds containing aluminium oxide NPs may present toxic effects after certain thresholds. Thus, the present study was carried out to determine the effects of Al2O3 nanoparticles (Al-NPs) in rats. For this aim, different doses (0, 0.5, 5, 50 mg/kg b.w./day) of Al NP (˜40 nm) were orally administered to female rats (Rattus norvegicus var. albinus) for 14 days and the response of several biomarkers such as activities of ATPases (total ATPase, Na,K-ATPase, Mg-ATPase) and acetylcholinesterase (AChE), levels of different glutathione forms and thiobarbituric acid reactive substances (TBARS) were measured in different tissues. Additionally, tissue accumulation of Al-NPs was demonstrated by a transmission electron microscope (TEM). The images showed the presence of Al-NP aggregates in all the tissues at all doses. The sizes of NP aggregates were dependent on NP doses and it was a bit more loose in the brain than in the liver and kidney. AChE activity in the brain decreased significantly at all NP doses, whereas TBARS levels in the liver did not alter significantly at any NP dose. Although there was no significant change in ATPase activities in the intestine at any NP dose, there were significant decreases in the kidney and brain. There were some variations in the levels of total glutathione (tGSH), oxidized glutathione (GSSG) and reduced glutathione (rGSH), though these variations were not significant (P > 0.05). Likewise, the ratio of rGSH/GSSG also did not differ significantly among NP doses and control. The brain seems most affected organ following Al-NP administration. This study demonstrated that most biomarkers in the tissues of rats were affected by Al-NP, showing the signal of toxic effects and suggests further studies to understand better the effects of Al NPs, especially in their use for pharmacology.


Assuntos
Óxido de Alumínio/toxicidade , Nanopartículas/toxicidade , Acetilcolinesterase/metabolismo , Adenosina Trifosfatases/metabolismo , Administração Oral , Animais , Biomarcadores/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Glutationa/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
13.
Nat Commun ; 10(1): 1720, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979890

RESUMO

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.


Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/análise , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Animais , Tampões (Química) , DNA Helicases/química , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Humanos , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Xenopus
14.
Plant Mol Biol ; 100(1-2): 163-179, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937701

RESUMO

KEY MESSAGE: We demonstrate that the C-terminus of OsCDC48 is essential for maintaining its full ATPase activity and OsCDC48/48E interaction is required to modulate cellular processes and plant survival in rice. Cell division cycle 48 (CDC48) belongs to the superfamily protein of ATPases associated with diverse cellular activities (AAA). We previously isolated a rice CDC48 mutant (psd128) displaying premature senescence and death phenotype. Here, we showed that OsCDC48 (Os03g0151800) interacted with OsCDC48E (Os10g0442600), a homologue of OsCDC48, to control plant survival in rice. OsCDC48E knockout plants exhibited similar behavior to psd128 with premature senescence and plant death. Removal of the C-terminus of OsCDC48 caused altered expression of cell cycle-related genes, changed the percentage of cells in G1 and G2/M phases, and abolished the interaction between OsCDC48 itself and between OsCDC48 and OsCDC48E, respectively. Furthermore, the truncated OsCDC48-PSD128 protein lacking the C-terminal 27 amino acid residues showed a decreased level of ATPase activity. Overexpression of OsCDC48-psd128 resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, accumulation of reactive oxygen species and decreased plant tiller numbers while overexpression of OsCDC48 also resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, but increased plant tiller numbers and grain yield, indicating its potential utilization for yield improvement. Our results demonstrated that the C-terminal region of OsCDC48 was essential for maintaining the full ATPase activity and OsCDC48/48E complex might function in form of heteromultimers to modulate cellular processes and plant survival in rice.


Assuntos
Oryza/fisiologia , Proteínas de Plantas/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Bases , Ciclo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Fenótipo , Desenvolvimento Vegetal , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Deleção de Sequência
15.
PLoS Genet ; 15(4): e1008065, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946745

RESUMO

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Domínio MADS/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Código das Histonas/genética , Proteínas de Domínio MADS/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Modelos Biológicos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Essays Biochem ; 63(1): 133-145, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30967477

RESUMO

The organization and regulation of genomic DNA as nuclear chromatin is necessary for proper DNA function inside living eukaryotic cells. While this has been extensively explored, no true consensus is currently reached regarding the exact mechanism of chromatin organization. The traditional view has assumed that the DNA is packaged into a hierarchy of structures inside the nucleus based on the regular 30-nm chromatin fiber. This is currently being challenged by the fluid-like model of the chromatin which views the chromatin as a dynamic structure based on the irregular 10-nm fiber. In this review, we focus on the recent progress in chromatin structure elucidation highlighting the paradigm shift in chromatin folding mechanism from the classical textbook perspective of the regularly folded chromatin to the more dynamic fluid-like perspective.


Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína
17.
Essays Biochem ; 63(1): 45-58, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30967479

RESUMO

ATP-dependent chromatin remodelling enzymes play a fundamental role in determining how nucleosomes are organised, and render DNA sequences accessible to interacting proteins, thereby enabling precise regulation of eukaryotic genes. Remodelers conserved from yeast to humans are classified into four families based on the domains and motifs present in their ATPase subunits. Insights into overall assembly and the mode of interaction to the nucleosome by these different families of remodelers remained limited due to the complexity of obtaining structural information on these challenging samples. Electron microscopy and single-particle methods have made advancement and uncovered vital structural information on the number of remodelling complexes. In this article, we highlight some of the recent structural work that advanced our understanding on the mechanisms and biological functions of these ATP-dependent remodelling machines.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Nucleossomos/metabolismo , Animais , DNA/metabolismo , Histonas/metabolismo , Humanos
18.
BMC Res Notes ; 12(1): 219, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971308

RESUMO

OBJECTIVES: Little is known about the activity and dynamics of ATPase RarA in B. subtilis, proposed to act at stalled DNA replication forks due to DNA damage. We performed fluorescence microscopy time lapse experiments with a functional RarA-mVenus fusion to visualize the dynamics of RarA during conditions that generate DNA damage. DATA DESCRIPTION: In exponentially growing cells, we observed that 15% of the cells contained single RarA-mV (mVenus fluorescent fusion) foci moving throughout the entire cell between 3 min intervals. This percentage remained constant at different time points, indicating that focus formation during unperturbed growth is maintained at about a constant rate. When cells were exposed to stress conditions, the population of cells containing RarA-mV foci tripled after 60 min. Cells exposed to two DNA-damaging drugs, to 5 mM MMS or to 0.5 mM H2O2, showed a similar type of response, with RarA-mVenus foci moving more slowly than during unperturbed growth. It is likely that RarA-mV contributes to the repair of H2O2-induced lesions, and to a minor extent to MMS-induced lesions. The presence of foci in growing cells suggests that RarA also plays a role during the cell cycle, at least in a fraction of cells, possibly contributing to heterogeneity of response to DNA damage.


Assuntos
Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Imagem com Lapso de Tempo
19.
Proc Natl Acad Sci U S A ; 116(12): 5533-5541, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30837315

RESUMO

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Microtúbulos/metabolismo , Espastina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Drosophila melanogaster/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(13): 6111-6119, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850548

RESUMO

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Catálise , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Ativação Enzimática , Polarização de Fluorescência , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética
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