Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27.302
Filtrar
1.
Nat Commun ; 11(1): 4580, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917864

RESUMO

Proteasomal machinery performs essential regulated protein degradation in eukaryotes. Classic proteasomes are symmetric, with a regulatory ATPase docked at each end of the cylindrical 20S. Asymmetric complexes are also present in cells, either with a single ATPase or with an ATPase and non-ATPase at two opposite ends. The mechanism that populates these different proteasomal complexes is unknown. Using archaea homologs, we construct asymmetric forms of proteasomes. We demonstrate that the gate conformation of the two opposite ends of 20S are coupled: binding one ATPase opens a gate locally, and also opens the opposite gate allosterically. Such allosteric coupling leads to cooperative binding of proteasomal ATPases to 20S and promotes formation of proteasomes symmetrically configured with two identical ATPases. It may also promote formation of asymmetric complexes with an ATPase and a non-ATPase at opposite ends. We propose that in eukaryotes a similar mechanism regulates the composition of the proteasomal population.


Assuntos
Archaea/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/metabolismo , Archaea/genética , Microscopia Crioeletrônica , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Thermoplasma/genética , Thermoplasma/metabolismo
2.
PLoS One ; 15(8): e0238452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866191

RESUMO

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.


Assuntos
Acremonium/metabolismo , Membrana Celular/metabolismo , Cefalosporinas/biossíntese , Cefalosporinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Meios de Cultura/metabolismo , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , beta-Lactamas/metabolismo
3.
PLoS Genet ; 16(8): e1008569, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810145

RESUMO

Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin-condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast.


Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
4.
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
5.
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
6.
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
7.
Nucleic Acids Res ; 48(14): 7991-8005, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32621607

RESUMO

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Geobacillus stearothermophilus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA , DNA Helicases/química , DNA Helicases/isolamento & purificação , Desoxirribonuclease I/química , Desoxirribonuclease I/isolamento & purificação
8.
Proc Natl Acad Sci U S A ; 117(31): 18608-18616, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690696

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.


Assuntos
Adenosina Trifosfatases , Reparo do DNA/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Genoma Fúngico/genética , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Proc Natl Acad Sci U S A ; 117(26): 14970-14977, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541053

RESUMO

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Transporte Proteico
16.
PLoS Comput Biol ; 16(6): e1007903, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584811

RESUMO

Nucleotides comprise small molecules that perform critical signaling roles in biological systems. Adenosine-based nucleotides, including adenosine tri-, di-, and mono-phosphate, are controlled through their rapid degradation by diphosphohydrolases and ecto-nucleotidases (NDAs). The interplay between nucleotide signaling and degradation is especially important in synapses formed between cells, which create signaling 'nanodomains'. Within these 'nanodomains', charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of nucleotidase activity. To quantify these factors, we utilized reaction-diffusion numerical simulations of 1) adenosine triphosphate (ATP) hydrolysis into adenosine monophosphate (AMP) and 2) AMP into adenosine (Ado) via two representative nucleotidases, CD39 and CD73. We evaluate these sequentially-coupled reactions in nanodomain geometries representative of extracellular synapses, within which we localize the nucleotidases. With this model, we find that 1) nucleotidase confinement reduces reaction rates relative to an open (bulk) system, 2) the rates of AMP and ADO formation are accelerated by restricting the diffusion of substrates away from the enzymes, and 3) nucleotidase co-localization and the presence of complementary (positive) charges to ATP enhance reaction rates, though the impact of these contributions on nucleotide pools depends on the degree to which the membrane competes for substrates. As a result, these contributions integratively control the relative concentrations and distributions of ATP and its metabolites within the junctional space. Altogether, our studies suggest that CD39 and CD73 nucleotidase activity within junctional spaces can exploit their confinement and favorable electrostatic interactions to finely control nucleotide signaling.


Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina/metabolismo , Nucleotídeos/metabolismo , Cinética , Transdução de Sinais , Propriedades de Superfície
17.
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
18.
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
19.
Mol Cell ; 79(1): 127-139.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32437639

RESUMO

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Hidrólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Mol Cell ; 79(1): 99-114.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445620

RESUMO

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA