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1.
Res Vet Sci ; 138: 30-38, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34091227

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and is devastating the swine industry. MARC-145 cells, an African green monkey kidney cell line, are sensitive to PRRSV-2, and are often used for in vitro studies on PRRSV-2. Preliminary research has shown that glycyrrhizin, an important active component extracted from traditional Chinese medicinal licorice, significantly inhibits the proliferation of PRRSV-2 in MARC-145 cells; however, the in-depth molecular mechanism remains unclear. By determining the cell growth cycle, this study found that PRRSV-2 infection first increased the content of G1-phase MARC-145 cells and then decreased the content of G1-phase cells. Moreover, glycyrrhizin affected the role of PRRSV-2 in regulating the cell cycle. Furthermore, PRRSV-2 had the highest proliferation titer in G0/G1-phase MARC-145 cells, and glycyrrhizin reduced the content of PRRSV-2 in synchronized MARC-145 cells. According to the results of ATPase detection, PRRSV-2 infection weakened the Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in MARC-145 cells, while glycyrrhizin significantly enhanced their activities in PRRSV-2-infected MARC-145 cells. The above results provide theoretical support toward clarifying the mechanism by which glycyrrhizin inhibits the proliferation of PRRSV-2 in MARC-145 cells. Moreover, these results offer references for the development and use of glycyrrhizin and the clinical treatment of PRRSV-2 infection.


Assuntos
Adenosina Trifosfatases/metabolismo , Antivirais/farmacologia , Ácido Glicirrízico/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Rim , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Vírus da Síndrome Respiratória e Reprodutiva Suína/enzimologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Suínos
2.
Nat Commun ; 12(1): 3312, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083526

RESUMO

Self-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we experimentally show that the Min protein dynamics on the membrane crucially depend on the micro chamber height due to bulk concentration gradients orthogonal to the membrane. A theoretical analysis shows that in vitro patterns at low microchamber height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger microchamber height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work reveals the qualitatively different mechanisms of mass transport that govern Min protein-patterns for different bulk heights and thus shows that Min patterns in cells are governed by a different mechanism than those in vitro.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Polaridade Celular , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Técnicas In Vitro , Modelos Biológicos , Dinâmica não Linear
3.
Nat Commun ; 12(1): 3483, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108481

RESUMO

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Adenosina Trifosfatases/química , Compostos de Boro/química , Proteínas de Saccharomyces cerevisiae/química , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Compostos de Boro/farmacologia , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Mutação , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Front Cell Infect Microbiol ; 11: 682635, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34150677

RESUMO

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.


Assuntos
Shigella , Sistemas de Secreção Tipo III , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Proteico , Shigella/metabolismo , Shigella flexneri/genética , Shigella flexneri/metabolismo , Sistemas de Secreção Tipo III/genética
5.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069632

RESUMO

In tobacco, the efficiency of Zn translocation to shoots depends on Zn/Cd status. Previous studies pointed to the specific contribution of root parts in the regulation of this process, as well as the role of NtZIP4A/B (from the ZIP family; Zrt Irt-like Proteins). Here, to verify this hypothesis, NtZIP4A/B RNAi lines were generated. Then, in plants exposed to combinations of Zn and Cd concentrations in the medium, the consequences of NtZIP4A/B suppression for the translocation of both metals were determined. Furthermore, the apical, middle, and basal root parts were examined for accumulation of both metals, for Zn localization (using Zinpyr-1), and for modifications of the expression pattern of ZIP genes. Our results confirmed the role of NtZIP4A/B in the control of Zn/Cd-status-dependent transfer of both metals to shoots. Furthermore, they indicated that the middle and basal root parts contributed to the regulation of this process by acting as a reservoir for excess Zn and Cd. Expression studies identified several candidate ZIP genes that interact with NtZIP4A/B in the root in regulating Zn and Cd translocation to the shoot, primarily NtZIP1-like in the basal root part and NtZIP2 in the middle one.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Tabaco/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico/genética , Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas/genética , Homeostase , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
6.
Nucleic Acids Res ; 49(11): 6474-6488, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34050764

RESUMO

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.


Assuntos
Adenosina Trifosfatases/química , Empacotamento do Genoma Viral , Proteínas Virais/química , Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Arginina/química , Fagos Bacilares/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Fosfatos/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Proteínas Virais/metabolismo
7.
Biochemistry (Mosc) ; 86(5): 540-550, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33993861

RESUMO

Ischemia/reperfusion (I/R) is among the most frequent neurological problems and early intervention to limit the damage is crucial in decreasing mortality and morbidity. Based on reports regarding beneficial effects of melatonin, we investigated its impact on Na+-K+/Mg2+ ATPase and Ca2+/Mg2+ ATPase activities and ultrastructure of gray and white matter in the rat forebrain I/R model. Adult Wistar-albino rats (n = 78), were randomized into control, ischemia (I), ischemia/reperfusion (I/R), low (I/R + melatonin 400 µg/kg), moderate (I/R + melatonin 1200 µg/kg), and high (I/R + melatonin 2400 µg/kg) dose melatonin. Two-vessel occlusion combined with hypotension (15 min) induced ischemia and reperfusion (75 min) achieved by blood reinfusion were performed. Activities of the membrane-bound enzyme, brain malondialdehyde levels, and brain matter ultrastructure were examined in frontoparietal cortices. Melatonin lowered production of malondialdehyde in a dose-dependently. The enzyme activities attenuated under I and I/R, improved with melatonin treatment. I and I/R severely disturbed gray and white matter morphology. Melatonin, in all applied doses, decreased ultrastructural damages in both gray and white matter. Favorable effects of melatonin can be attributed to its antioxidant properties suggesting that it could be a promising neuroprotective agent against I/R injury being effective both for gray and white matter due to favorable biological properties.


Assuntos
Adenosina Trifosfatases/metabolismo , Substância Cinzenta/enzimologia , Melatonina/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Substância Branca/enzimologia , Animais , Isquemia Encefálica , Modelos Animais de Doenças , Substância Cinzenta/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Substância Branca/metabolismo
8.
Pestic Biochem Physiol ; 175: 104833, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33993958

RESUMO

Neurochemical and ATPase deregulations play important role in toxicant-induced neurodegeneration. Previous studies have shown that loss of ATPase ionic-pumps alters neurochemical balance via increased ammonia, oxidative and nitrosative stress. Thus, this study investigated the ameliorative potentials of quercetin on neurochemical, ATPase changes, hyperammonemia and oxidative/nitrosative status in the brains of Wistar rats exposed to endosulfan, a known toxic environmental pesticide that is casually used in many developing countries. Adult rats were divided into five treatment groups (n = 5). Groups 1-2 received normal saline and corn oil (vehicle) (10 mL/kg/day), group 3 received quercetin (20 mg/kg/day) orally for 28 days consecutively. However, animals in groups 4-5 were given endosulfan (5 mg/kg/day, p.o) for 28 days. But, from the 14th to 28th day, group 4 additionally received vehicle (10 mL/kg/day, p.o.), while group 5 was treated with quercetin (20 mg/kg/day, p.o.). Thereafter, brain levels of neurochemicals, ATPase activities, ammonia and oxidative/nitrosative stress were investigated by employing standardized biochemical assay protocols. Quercetin increased endosulfan-induced decreased levels of norepinephrine, dopamine, GABA, and decreased elevated concentrations of glutamate and serotonin. Quercetin normalized the increased levels of acetylcholinesterase and ammonia. Furthermore, quercetin significantly reversed the decrease in Na+/K+, Ca2+, Mg2+-ATPase activities induced by endosulfan. Also, quercetin increased superoxide dismutase, catalase and glutathione peroxidase activities, and reduced nitrite and peroxynitrite levels in brains of rats. These findings further provide evidence of the ameliorative potential of quercetin against endosulfan-induced neurotoxicity via attenuation of neurochemical, ATPase changes, and inhibition of acetylcholinesterase activity, ammonia release and oxidative/nitrosative stress in rat brains.


Assuntos
Estresse Nitrosativo , Quercetina , Adenosina Trifosfatases/metabolismo , Animais , Antioxidantes , Encéfalo/metabolismo , Catalase/metabolismo , Endossulfano/toxicidade , Estresse Oxidativo , Quercetina/farmacologia , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo
9.
Vet Microbiol ; 257: 109074, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940460

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg2+ and Mn2+ were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing antiviral drugs.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Vírus da Diarreia Epidêmica Suína/enzimologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , RNA Viral/genética , RNA Viral/metabolismo , Suínos , Doenças dos Suínos/virologia , Células Vero
10.
Nihon Yakurigaku Zasshi ; 156(3): 166-170, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33952846

RESUMO

In the brains of patients with Alzheimer's disease, a decrease in phosphatidylinositol phosphate (PIP) requiring Cl--ATPase activity was found. In cultured rat hippocampal neurons, pathophysiological concentrations of amyloid ß proteins (Aßs≤10 nM) lowered PIP levels and Cl--ATPase activity with an increase in intracellular Cl- concentrations, resulting in Cl--dependent enhancements in glutamate neurotoxicity and, ultimately, neuronal cell death. Pathophysiological concentrations of Aßs(0.1-10 nM) directly lowered phosphatidylinositol-4-kinase. Non-toxic peptide fragments of Aß, such as Ile-Gly-Leu, recovered Aß-induced inhibition of recombinant human phosphatidylinositol-4-kinase IIα (PI4KIIα) and the intrahippocampally administered Aß-induced degeneration of hippocampal neurons and impairment of spatial memory in mice. Agents with the potential to block these neurotoxic mechanisms of Aß were summarized herein as (1) Aß antagonists, (2) substrates of PI4K, (3) PI4K product, (4) PI4K activators, and (5) GABAc receptor stimulants.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Adenosina Trifosfatases/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Morte Celular , Células Cultivadas , Hipocampo/metabolismo , Humanos , Camundongos , Fragmentos de Peptídeos , Ratos , Ratos Wistar
11.
Sci Transl Med ; 13(587)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790022

RESUMO

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Adenosina Trifosfatases/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteína com Valosina
12.
Nucleic Acids Res ; 49(8): 4534-4549, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849072

RESUMO

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/metabolismo , Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
13.
Food Chem ; 356: 129704, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33831827

RESUMO

The postharvest senescence accompanied by yellowing limited the shelf-life of broccoli. In this study, we developed a novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil and applied it to broccoli for preservation. Results showed that double emulsion prepared by whey protein concentrate-high methoxyl pectin (1:3) exhibited best storage stability with largest particle size (581.30 nm), lowest PDI (0.23) and zeta potential (-40.31 mV). This double emulsion also exhibited highest encapsulation efficiency of brassinolide (92%) and cinnamon essential oil (88%). The broccoli coated with double emulsion maintained higher chlorophyll contents and activities of chlorophyllase and magnesium-dechelatase were reduced by 9% and 24%, respectively. The energy metabolic enzymes (SDH, CCO, H+-ATPase, Ca2+-ATPase) were also activated, inducing higher level of ATP and energy charge. These results demonstrated W/O/W double emulsion co-delivering brassinolide and cinnamon essential delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.


Assuntos
Brassica/metabolismo , Brassinosteroides/química , Clorofila/metabolismo , Emulsões/química , Metabolismo Energético , Óleos Voláteis/química , Esteroides Heterocíclicos/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Brassica/efeitos dos fármacos , Brassinosteroides/metabolismo , Brassinosteroides/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Cinnamomum zeylanicum/metabolismo , Emulsões/metabolismo , Metabolismo Energético/efeitos dos fármacos , Enzimas/química , Armazenamento de Alimentos/métodos , Óleos Voláteis/metabolismo , Óleos Voláteis/farmacologia , Tamanho da Partícula , Esteroides Heterocíclicos/metabolismo , Esteroides Heterocíclicos/farmacologia , Viscosidade
14.
Methods Mol Biol ; 2263: 289-318, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877604

RESUMO

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a single turnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.


Assuntos
Cumarínicos/química , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Nucleotídeos/química , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/análise , Adenosina Trifosfatases/metabolismo , Técnicas Biossensoriais , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrólise , Cinética , Proteínas de Ligação a Fosfato/química
15.
Arch Biochem Biophys ; 703: 108841, 2021 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-33775623

RESUMO

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Leishmania major/enzimologia , Multimerização Proteica , Sequência de Aminoácidos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Soluções
16.
Nat Commun ; 12(1): 1488, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674615

RESUMO

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.


Assuntos
Éxons , RNA Helicases/química , RNA Helicases/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , Precursores de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Fúngico/metabolismo , Proteínas Recombinantes , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/química
17.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670267

RESUMO

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Deleção de Sequência
18.
Biomed Res Int ; 2021: 4615384, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604374

RESUMO

Proton pumps are membrane-bound enzymes important in generating gradients that help in maintaining cellular ion homeostasis, cell membrane potential, water, and solute transport across the cell surface. This study investigated the modulatory role of vitamin E on proton pump activity and reproductive parameters in cadmium-induced testicular damage. Twenty (20) male Wistar rats weighing between 180 and 200 g were sorted into 4 groups of five rats each. Group I served as the control and was given normal saline orally, Group II rats were treated with a single dose of 2 mg/kg BW cadmium chloride (CdCl2) intraperitoneally, Group III rats were given 100 mg/kg BW of vitamin E orally, and Group IV rats were given 100 mg/kg BW of vitamin E orally for 30 days prior to intraperitoneal administration of single dose of 2 mg/kg BW of cadmium chloride. The rats were anaesthetized with diethyl ether, and blood samples were obtained for sex hormonal analysis; caudal epididymis was dissected for sperm count, motility, and viability, and the testis were homogenized for lipid peroxidation and proton pump (Na+/K+ ATPase, Ca2+ ATPase, and Mg2+ ATPase) activity. Proton pump activity was assayed spectrophotometrically using the Stewart method to determine the inorganic phosphate level. Histopathological changes of the testis were also studied. The group treated with CdCl2 showed a significant (p < 0.05) decrease in proton pump activity, sperm count, and motility and a significant (p < 0.05) increase in malondialdehyde level when compared with the control group. The CdCl2-treated group also showed decrease reproductive organ weights and hormonal levels and cause necrosis of spermatogonia lining the seminiferous tubules. Rats treated with vitamin E orally for 30 days prior to CdCl2 exposure showed improvement in proton pump activity, a significant (p < 0.05) increase in sperm parameters and luteinizing hormonal level, and a decrease in the lipid peroxidation level as compared with the CdCl2 group. This study showed that vitamin E ameliorated the toxic effect of CdCl2 on proton pump activity in the testes, hence improving testicular integrity, structures, and functions.


Assuntos
Adenosina Trifosfatases , Cloreto de Cádmio/efeitos adversos , Bombas de Próton , Testículo , Vitamina E/farmacologia , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Bombas de Próton/efeitos dos fármacos , Bombas de Próton/metabolismo , Ratos , Ratos Wistar , Doenças Testiculares/induzido quimicamente , Doenças Testiculares/metabolismo , Testículo/efeitos dos fármacos , Testículo/enzimologia , Testículo/metabolismo , Testículo/patologia
19.
Nat Commun ; 12(1): 782, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542241

RESUMO

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Mutação , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Partícula de Reconhecimento de Sinal/metabolismo , Ubiquitina/genética , Ubiquitina/isolamento & purificação
20.
Nucleic Acids Res ; 49(4): 2161-2178, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533920

RESUMO

Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.


Assuntos
Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Staphylococcus aureus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA/química , Farmacorresistência Bacteriana , Transferência Genética Horizontal , Modelos Moleculares , Multimerização Proteica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
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