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1.
PLoS Genet ; 16(7): e1008904, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730253

RESUMO

The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds ATP but cannot hydrolyze it: pch-2E253Q. In vitro, this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2's adapter protein, CMT-1, the ortholog of p31comet, localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Meiose/genética , Proteínas Nucleares/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Animais , Caenorhabditis elegans/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Humanos , Mutação/genética , Fuso Acromático/genética
2.
Nat Commun ; 11(1): 3405, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636378

RESUMO

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.


Assuntos
Miosinas Cardíacas/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miosinas/metabolismo , Fosfatos/farmacologia , Ureia/análogos & derivados , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Sinergismo Farmacológico , Masculino , Músculo Esquelético/metabolismo , Coelhos , Sarcômeros/metabolismo , Estresse Mecânico , Ureia/farmacologia
3.
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
4.
Proc Natl Acad Sci U S A ; 117(30): 17775-17784, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669440

RESUMO

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , DNA/química , DNA/genética , Hidrólise , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas MutL/química , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Relação Estrutura-Atividade
5.
PLoS One ; 15(7): e0235033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639961

RESUMO

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


Assuntos
Adenosina Trifosfatases/genética , Antimaníacos/farmacologia , Cloreto de Lítio/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fosfoglucomutase/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
6.
PLoS Pathog ; 16(7): e1008709, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730331

RESUMO

Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting. In vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.


Assuntos
Proteínas do Movimento Viral em Plantas/metabolismo , Vírus de Plantas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Tabaco/virologia , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/fisiologia , Adenosina Trifosfatases/metabolismo , Potexvirus/fisiologia , Ribonucleoproteínas/metabolismo
7.
Mol Cell ; 79(1): 3-5, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619470

RESUMO

Kong et al. (2020) present the low-resolution structure of the ATPÉ£S-bound human condensin I and II complexes and demonstrate that human condensins can extrude DNA loops in a symmetric and asymmetric fashion and compact nucleosome-bound DNA.


Assuntos
Adenosina Trifosfatases/genética , Nucleossomos , Trifosfato de Adenosina , DNA , Proteínas de Ligação a DNA , Humanos , Complexos Multiproteicos
8.
No Shinkei Geka ; 48(6): 533-540, 2020 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-32572006

RESUMO

Aplastic or twig-like middle cerebral artery(Ap/T-MCA)is a rare congenital anomaly, and several cases of ruptured cerebral aneurysm associated with Ap/T-MCA have been reported. Recently, the association of ring finger protein 213(RNF213)mutations with moyamoya disease has been identified, and the involvement of such mutations in intracranial arterial stenosis lesions other than those of moyamoya disease has been suggested. A 53-year-old woman with headache and nausea was admitted to our hospital. Computed tomography showed a diffuse subarachnoid hemorrhage. Cerebral angiography revealed left-sided Ap/T-MCA and two aneurysms in several fine arterioles. We performed trapping of these aneurysms. In the clinical course after surgery, she developed aphasia and mild motor paralysis. The patient was transferred to a rehabilitation hospital. The genetic screening revealed that she carried a heterozygous mutation of RNF213(c. 14429G>A p. R4810K). This is the first report of an association between Ap/T-MCA and RNF213 mutations. In patients with the RNF213 mutation, there is also the possibility of a progression of the intracranial arterial stenosis to other sites. Such patients should be carefully observed after the completion of their treatment.


Assuntos
Aneurisma Roto , Aneurisma Intracraniano , Doença de Moyamoya , Adenosina Trifosfatases/genética , Angiografia Cerebral , Feminino , Humanos , Pessoa de Meia-Idade , Artéria Cerebral Média , Mutação , Ubiquitina-Proteína Ligases/genética
9.
Ecotoxicol Environ Saf ; 201: 110725, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32474209

RESUMO

Lincomycin hydrochloride is one of the commonly used drugs in clinic. However, it has many side effects on patients, and its mechanism is still poorly understood. In this study, 6 h post-fertilization (6 hpf) zebrafish embryos were exposed to several concentrations of lincomycin hydrochloride (15, 30, 60 µg/mL) for up to 24 or 96 hpf to detect their developmental toxicity and neurotoxicity, and to 6 days post-fertilization (6 dpf) to detect their behavioral toxicity. Our results showed that lincomycin hydrochloride could lead to embryonic head deformities (unclear ventricles, smaller ventricles, fewer new neurons). The studies showed that the frequency of spontaneous tail flick of zebrafish embryo increased at 24 hpf, and the lincomycin hydrochloride exposed zebrafish embryos showed increased heart rate, shorter body length, and yolk sac edema with severe pericardial edema at 96 hpf. The studies also showed that lincomycin hydrochloride increased oxidative stress level, Acetylcholinesterase (AChE) activity, ATPase activity and apoptosis in zebrafish larvae. In addition, the swimming behavior of zebrafish larvae decreased with the increase of lincomycin hydrochloride concentration, but the angular velocity and meandering degree increased, which might be due to the decreased activity of AChE and ATPase, as well as the decreased expression of genes related to neurodevelopment and neurotransmitter system, leading to the change of their motor behaviors. In summary, we found that lincomycin hydrochloride induced developmental toxicity and neurotoxicity in zebrafish larvae, contributing to a more comprehensive evaluation of the safety of the drug.


Assuntos
Lincomicina/toxicidade , Síndromes Neurotóxicas/etiologia , Acetilcolinesterase/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Síndromes Neurotóxicas/congênito , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra
10.
J Environ Sci Health B ; 55(8): 749-755, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32558613

RESUMO

This study assessed the hematological, enzymatic and osmoregulatory responses of silver catfish (Rhamdia quelen) exposed to sublethal concentrations (1.125 and 3.750 µg/L) of a commercial thiamethoxam-containing insecticide used on rice crops. Groups of 6 fish per tank (in triplicate, n = 3, total 54 fish) were exposed for up to 96 h to different concentrations of the compound. After this period, fish were placed in clean water for 48 h. Two fish from each tank (6 per treatment) that had been exposed to the insecticide for 24 h were anesthetized with eugenol and blood was collected to evaluate hematological and biochemical parameters. Blood, liver and muscle were collected for determination of metabolic parameters, plasma cortisol, Cl-, Na+ and K+ levels and H+-ATPase and Na+/K+-ATPase activity in the gill. H+-ATPase activity was higher in fish exposed to 1.125 µg/L insecticide at 24 h compared to control (0.0 µg/L). Differences in cortisol levels were evidenced throughout the experimental period. These results indicated that exposure to the insecticide changed the hematological, biochemical and metabolic profile of the animals, suggesting concern about environmental safety. Therefore, we discourage the use of this pesticide in areas that come into contact with water bodies inhabited by fish.


Assuntos
Peixes-Gato/fisiologia , Inseticidas/toxicidade , Tiametoxam/toxicidade , Adenosina Trifosfatases/metabolismo , Animais , Peixes-Gato/sangue , Ecotoxicologia/métodos , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hidrocortisona/sangue , Fígado/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade
11.
Virol Sin ; 35(3): 321-329, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32500504

RESUMO

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.


Assuntos
Betacoronavirus/metabolismo , Bismuto/farmacologia , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/efeitos dos fármacos , RNA Helicases/efeitos dos fármacos , Sais/farmacologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Betacoronavirus/enzimologia , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes , Síndrome Respiratória Aguda Grave , Proteínas não Estruturais Virais/genética , Replicação Viral
12.
Toxicol Appl Pharmacol ; 401: 115080, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32497533

RESUMO

Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more "patient-specific" LCL161 clinical trial program in the future.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Mitocondriais/farmacologia , Survivina/antagonistas & inibidores , Tiazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Proteínas Reguladoras de Apoptose/química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Mitocondriais/química , Estrutura Secundária de Proteína , Survivina/biossíntese , Survivina/genética , Tiazóis/química
13.
Nat Commun ; 11(1): 2728, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483114

RESUMO

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Conformação Proteica , Multimerização Proteica , Transativadores/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenol/metabolismo , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo
14.
Nat Commun ; 11(1): 2818, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499524

RESUMO

In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. Here, we show that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.


Assuntos
Adenosina Trifosfatases/metabolismo , Autoantígenos/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/química , Histona Desacetilase 1/metabolismo , Nucleossomos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Drosophila melanogaster , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Histonas/química , Ovário/metabolismo , Proteínas Inibidoras de STAT Ativados
15.
Nat Commun ; 11(1): 2887, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513971

RESUMO

In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Histonas/genética , Chaperonas Moleculares/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Sequenciamento Completo do Genoma/métodos
16.
Life Sci ; 256: 117862, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32473244

RESUMO

Vascular smooth muscle cells (VSMCs) exhibit a high degree of plasticity when they undergo the progression from a normal to a disease condition, which makes them a potential target for evaluating early markers and for the development of new therapies. Purinergic signalling plays a key role in vascular tonus control, ATP being an inductor of vasoconstriction, whereas adenosine mediates a vasodilation effect antagonising the ATP actions. The control of extracellular ATP and adenosine levels is done by ectonucleotidases, which represent a potential target to be evaluated in the progression of cardiovascular diseases. In this study, we analysed the basal activity and expression of the ectonucleotidases in aortic rat VSMCs, and we further performed in silico analysis to determine the expression of those enzymes in conditions that mimicked vascular diseases. Cultured in vitro VSMCs showed a prominent expression of Entpd1 followed by Entpd2 and Nt5e (CD73) and very low levels of Entpd3. Slightly faster AMP hydrolysis was observed when compared to ATP and ADP nucleotides. In silico analysis showed that the ectonucleotidases were modulated after induction of conditions that can lead to vascular diseases such as, hypertensive and hypotensive mice models (Nt5e); exposition to high-fat (Entpd1 and Entpd2) or high-phosphate (Nt5e) diet; mechanical stretch (Entpd1, Entpd2 and Nt5e); and myocardial infarction (Entpd1). Our data show that VSMCs are able to efficiently metabolise the extracellular nucleotides generating adenosine. The modulation of Entpd1, Entdp2 and Nt5e in vascular diseases suggests these ectoenzymes as potential targets or markers to be investigated in future studies.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Músculo Liso Vascular/patologia , Doenças Vasculares/fisiopatologia , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Simulação por Computador , Proteínas Ligadas por GPI/metabolismo , Camundongos , Músculo Liso Vascular/enzimologia , Nucleotídeos/metabolismo , Ratos , Ratos Wistar , Doenças Vasculares/enzimologia
17.
Nucleic Acids Res ; 48(11): 6326-6339, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374860

RESUMO

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Soluções , Eletricidade Estática , Sulfolobus/química
18.
J Biosci Bioeng ; 130(2): 149-158, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32414665

RESUMO

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Regulação da Expressão Gênica em Archaea , Hidrogênio/metabolismo , Thermococcus/enzimologia , Thermococcus/genética , Carbono/metabolismo , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica em Archaea/genética , Organismos Geneticamente Modificados/metabolismo , Polissacarídeos/metabolismo , Ácido Pirúvico/metabolismo
19.
Pol J Pathol ; 71(1): 69-74, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32429658

RESUMO

Molecular next gene sequencing was used to evaluate mutations in 409 common mutated cancer-related genes in malignant mesothelioma of tunica vaginalis testis (MMTVT) of 81-year-old man. Multifocal papillary-solid areas contained necrosis among highly cellular fields with multiple mitoses. It was positive for WT1, CKAE1/AE3, calretinin, CK7 with negativity for CK5, PSA, TTF-1. Following mutations were revealed in PARP1 (NM_001618: c.2285TG, p.K135R), MTRR (NM_024010: c.147A>G, p.I49M) and two sorts of mutations in structure of KMT2C gene (NM_170606: c.2447_2448insA (c.2447dupA), p.Y816fs and NM_170606: c.1042G>A, p.D348N) for the first time in MMTVT.


Assuntos
Mesotelioma/genética , Neoplasias Testiculares/genética , Adenosina Trifosfatases/genética , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Ferredoxina-NADP Redutase/genética , Humanos , Masculino , Mutação , Fator de Transcrição PAX8/genética , Poli(ADP-Ribose) Polimerase-1/genética , Ubiquitina-Proteína Ligases/genética
20.
Nucleic Acids Res ; 48(12): 6980-6995, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453425

RESUMO

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/ultraestrutura , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Conformação Proteica
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