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1.
J Agric Food Chem ; 68(10): 3006-3016, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-31986035

RESUMO

An increase in crop competitiveness relative to weed interference has the potential to reduce crop yield losses. In this study, the effects of phytoalexin resveratrol were examined in Zea mays L. (corn) and in the weed species Ipomoea grandifolia (Dammer) O'Donell (morning glory). At a concentration range from 220 to 2200 µM resveratrol exerted a stimulus on Z. mays seedling growth that was more pronounced at low concentrations; in the weed species I. grandifolia, resveratrol exerted inhibitory action on seedling growth in all of the assayed concentration range. In I. grandifolia, resveratrol also inhibited the respiratory activity of the primary roots. In mitochondria isolated from Z. mays roots, resveratrol at concentrations above 440 µM inhibited the respiration coupled to ADP phosphorylation and the activities of NADH-oxidase, succinate-oxidase, and ATPsynthase. These effects were not reproduced in Z. mays grown in the presence of resveratrol as the respiratory activities of the roots were not affected. The finding that the resveratrol exerts beneficial effects on growth of Z. mays seedlings and inhibits the growth of I. grandifolia heightens the potential of resveratrol application for crop protection.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Ipomoea/efeitos dos fármacos , Resveratrol/farmacologia , Zea mays/efeitos dos fármacos , Ipomoea/crescimento & desenvolvimento , Ipomoea/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/crescimento & desenvolvimento , Plantas Daninhas/metabolismo , Resveratrol/análise , Sesquiterpenos/análise , Sesquiterpenos/farmacologia , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
2.
Biochim Biophys Acta Bioenerg ; 1861(1): 148118, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734195

RESUMO

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH-. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.


Assuntos
Aldeído Oxirredutases/química , Bradyrhizobiaceae/enzimologia , Metaloproteínas/química , Complexos Multienzimáticos/química , Aldeído Oxirredutases/metabolismo , Catálise , Domínio Catalítico , Cobalto/química , Cobalto/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Metaloproteínas/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Complexos Multienzimáticos/metabolismo
3.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1870-1888, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668035

RESUMO

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.


Assuntos
Biotecnologia , Enzimas/química , Enzimas/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Biologia Sintética
4.
Enzymes ; 45: 183-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627877

RESUMO

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Complexos Multienzimáticos/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/química
5.
BMC Res Notes ; 12(1): 587, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533811

RESUMO

OBJECTIVE: Fetal macrosomia is a major risk factor for shoulder dystocia, which can lead to birth asphyxia, maternal and neonatal traumatic injuries, and perinatal death. If macrosomia is diagnosed in the antenatal period, labour can be induced to decrease shoulder dystocia. But current clinical methods to diagnose fetal macrosomia antenatally perform with poor accuracy. Therefore, improved methods to accurately diagnose fetal macrosomia are required. Blood biomarkers that predict fetal macrosomia could be one such novel diagnostic strategy. We undertook a nested case-control study from a prospective collection of 1000 blood samples collected at 36 weeks' gestation. We analysed plasma samples from 52 women who subsequently delivered a macrosomic (> 95th centile for gestational age) infant and 106 controls. Circulating concentrations of the proteins COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 were assessed for their ability to predict macrosomic infants. RESULTS: We did not identify any significant changes in the plasma concentrations of COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 from women who subsequently delivered macrosomic neonates relative to control samples. Although we have not identified any potential biomarkers of fetal macrosomia, we have ruled out these particular eight protein candidates.


Assuntos
Biomarcadores/sangue , Macrossomia Fetal/sangue , Diagnóstico Pré-Natal/métodos , Proteínas/isolamento & purificação , Adulto , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/metabolismo , Feminino , Macrossomia Fetal/diagnóstico , Humanos , Recém-Nascido , Complexos Multienzimáticos/sangue , Complexos Multienzimáticos/metabolismo , Gravidez , Progesterona Redutase/sangue , Progesterona Redutase/metabolismo , Estudos Prospectivos , Proteínas/metabolismo , Sensibilidade e Especificidade , Esteroide Isomerases/sangue , Esteroide Isomerases/metabolismo , Fatores de Transcrição/sangue , Fatores de Transcrição/metabolismo
6.
Nat Struct Mol Biol ; 26(9): 830-839, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31488907

RESUMO

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.


Assuntos
Domínio Catalítico , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Precursores de RNA/metabolismo , Chaetomium/enzimologia , Microscopia Crioeletrônica , Conformação Proteica
7.
Prep Biochem Biotechnol ; 49(10): 949-960, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398070

RESUMO

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential application of these enzymes in the extraction of lycopene from tomato skin, waste, and paste (SWP). The CWDE was then immobilized via entrapment in alginate beads for lycopene extraction and the kinetic/thermodynamic properties of the free and immobilized CWDE investigated. The optimum production of CWDE occurred at pH, temperature, and orange peel concentration of 6.0, 50 °C, and 2.0% (w/v), respectively. The values obtained for some kinetic and thermodynamic parameters such as Ed*, t1/2, ΔGd*, and ΔHd* indicate that both free and immobilized cellulase and pectinase were thermostable between 40 and 50 °C. Maximum lycopene extracted from the tomato SWP was 80 ± 2.4 mg/kg, 42 ± 1.3 mg/kg and 60 ± 1.2 mg/kg, respectively, using the immobilized CWDE. The entrapped CWDE was able to extract lycopene with yields of 58 ± 4.2, 51 ± 1.2 and 57 ± 4.2% for tomato SWP respectively after the fifth cycle. Using orange peels for the induction of CWDE by A. pullulans offers a unique and cheaper approach to obtaining thermostable multi-enzyme complexes employable for easy lycopene extraction from tomato SWP.


Assuntos
Ascomicetos/enzimologia , Parede Celular/metabolismo , Celulase/metabolismo , Citrus sinensis , Enzimas Imobilizadas/metabolismo , Licopeno/isolamento & purificação , Poligalacturonase/metabolismo , Celulase/biossíntese , Concentração de Íons de Hidrogênio , Cinética , Complexos Multienzimáticos/metabolismo , Poligalacturonase/biossíntese , Termodinâmica
8.
Int J Med Microbiol ; 309(6): 151334, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31383542

RESUMO

The bacterial cell wall provides structural integrity to the cell and protects the cell from internal pressure and the external environment. During the course of the twelve-year funding period of the Collaborative Research Center 766, our work has focused on conducting structure-function studies of enzymes that modify (synthesize or cleave) cell wall components of a range of bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Nostoc punctiforme. Several of our structures represent promising targets for interference. In this review, we highlight a recent structure-function analysis of an enzyme complex that is responsible for the amidation of Lipid II, a peptidoglycan precursor, in S. aureus.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Peptidoglicano/metabolismo , Parede Celular/enzimologia , Parede Celular/metabolismo , Peptidoglicano/química , Domínios Proteicos , Staphylococcus/enzimologia , Staphylococcus/metabolismo , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
9.
Biochim Biophys Acta Bioenerg ; 1860(9): 734-744, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376363

RESUMO

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (N2O) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO2 and reducing nitrite to N2 without producing N2O. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O2 and N2 by a putative NO dismutase (NO-D). The O2 produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.


Assuntos
Bactérias Anaeróbias/enzimologia , Proteínas de Bactérias/metabolismo , Metano/química , Complexos Multienzimáticos/metabolismo , Nitratos/química , Óxido Nítrico/química , Metano/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Oxigenases/metabolismo
10.
Int J Mol Sci ; 20(14)2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31373299

RESUMO

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.


Assuntos
Biodegradação Ambiental , Dioxigenases/metabolismo , Poluentes Ambientais/metabolismo , Complexos Multienzimáticos/metabolismo , Naftalenos/metabolismo , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Ditionita/química , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Oxirredução
11.
J Enzyme Inhib Med Chem ; 34(1): 1439-1450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409157

RESUMO

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismo
12.
Anim Reprod Sci ; 208: 106110, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405469

RESUMO

Adiponectin is an adipocyte derived cytokine implicated in energy homeostasis, insulin resistance and is involved in the regulation of reproduction both centrally and peripherally in animals. The present study was conducted to investigate adiponectin (ADIPOQ) and its receptors ADIPOR1 and ADIPOR2 abundance of mRNA transcript and protein in different stages of corpora lutea (CL) development during the estrous cycle of water buffalo and to determine the effect of adiponectin on cultured luteal cells of water buffalo (Bubalus bubalis). The results indicate adiponectin, ADIPOR1, and ADIPOR2 were present in buffalo corpora lutea (CL) throughout the estrous cycle. The abundance of adiponectin and its receptors was greater in the early and regressing and was less in mid- and late-stages of CL functionality. Adiponectin and its receptors were localized in the cytoplasm of small and large luteal cells. Furthermore, luteal cells were cultured in the in-vitro culture system and were treated with 1 and 10 µg/mL dose of adiponectin for 48 h. Adiponectin at both doses decreased (P < 0.05) progesterone (P4) secretion from cultured luteal cells and also suppressed the abundance of factors involved in P4productionv [Steroidogenic Acute Regulatory Protein (STAR), cytochrome P45011A1 (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at the 10 µg/mL dose as compared to adiponectin non-supplemented cells]. In conclusion, results of the present study indicate adiponectin and its receptors are present in bubaline CL and adiponectin inhibits P4 production in cultured luteal cells. The findings indicate adiponectin affects luteal dynamics and reproductive functions in water buffalo.


Assuntos
Adiponectina/metabolismo , Búfalos/fisiologia , Corpo Lúteo/metabolismo , Ciclo Estral/fisiologia , Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/metabolismo , Adiponectina/genética , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Imuno-Histoquímica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Progesterona/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , RNA Mensageiro/genética , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismo
13.
J Biotechnol ; 304: 31-37, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31421146

RESUMO

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.


Assuntos
Butileno Glicóis/metabolismo , Deleção de Genes , Glicerolfosfato Desidrogenase/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Candida tropicalis/enzimologia , Fermentação , Proteínas Fúngicas/genética , Engenharia Genética , Glicerol/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Lactococcus lactis/enzimologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Piruvato Descarboxilase/deficiência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
14.
Nat Commun ; 10(1): 3005, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285450

RESUMO

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.


Assuntos
Complexos Multienzimáticos/ultraestrutura , Fosfoproteínas/ultraestrutura , Proteínas Serina-Treonina Quinases/ultraestrutura , Estresse Fisiológico , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Listeria monocytogenes/fisiologia , Complexos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Fator sigma/metabolismo , Transdução de Sinais/fisiologia
15.
Nat Struct Mol Biol ; 26(8): 704-711, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285605

RESUMO

In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharomyces cerevisiae Pmt1-Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal ß-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.


Assuntos
Manosiltransferases/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Glicosilação , Humanos , Processamento de Imagem Assistida por Computador , Manose/metabolismo , Manosiltransferases/química , Manosiltransferases/genética , Manosiltransferases/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Síndrome de Walker-Warburg/genética
16.
Nucleic Acids Res ; 47(16): 8662-8674, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31287872

RESUMO

A typical feature of eukaryotic aminoacyl-tRNA synthetases (aaRSs) is the evolutionary gain of domains at either the N- or C-terminus, which frequently mediating protein-protein interaction. TARSL2 (mouse Tarsl2), encoding a threonyl-tRNA synthetase-like protein (ThrRS-L), is a recently identified aaRS-duplicated gene in higher eukaryotes, with canonical functions in vitro, which exhibits a different N-terminal extension (N-extension) from TARS (encoding ThrRS). We found the first half of the N-extension of human ThrRS-L (hThrRS-L) is homologous to that of human arginyl-tRNA synthetase. Using the N-extension as a probe in a yeast two-hybrid screening, AIMP1/p43 was identified as an interactor with hThrRS-L. We showed that ThrRS-L is a novel component of the mammalian multiple tRNA synthetase complex (MSC), and is reliant on two leucine zippers in the N-extension for MSC-incorporation in humans, and mouse cell lines and muscle tissue. The N-extension was sufficient to target a foreign protein into the MSC. The results from a Tarsl2-deleted cell line showed that it does not mediate MSC integrity. The effect of phosphorylation at various sites of hThrRS-L on its MSC-targeting is also explored. In summary, we revealed that ThrRS-L is a bona fide component of the MSC, which is mediated by a newly evolved N-extension domain.


Assuntos
Arginina-tRNA Ligase/genética , Citocinas/genética , Complexos Multienzimáticos/genética , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Treonina-tRNA Ligase/genética , Sequência de Aminoácidos , Animais , Arginina-tRNA Ligase/metabolismo , Clonagem Molecular , Citocinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Zíper de Leucina , Camundongos , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Treonina-tRNA Ligase/metabolismo , Técnicas do Sistema de Duplo-Híbrido
17.
PLoS One ; 14(7): e0219524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291327

RESUMO

OBJECTIVES: The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size. RESULTS: Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis.


Assuntos
Células Intersticiais do Testículo , Complexos Multienzimáticos/genética , Progesterona Redutase/genética , Esteroide Isomerases/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Contagem de Células/métodos , Perfilação da Expressão Gênica , Masculino , Camundongos , Complexos Multienzimáticos/metabolismo , Progesterona Redutase/metabolismo , Esteroide Isomerases/metabolismo
18.
PLoS One ; 14(7): e0220098, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329647

RESUMO

Curcuma is a traditional ingredient of some Eastern cuisines, and the spice is heralded for its antitumoral and antiparasitic properties. In this report, we examine the effect of the curcuminoides which include curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), as well as curcumin degradation products on thioredoxin glutathione reductase from Taenia crassiceps cysticerci Results revealed that both DMC and BDMC were inhibitors of TGR activity in the micromolar concentration range. By contrast, the inhibitory ability of curcumin was a time-dependent process. Kinetic and spectroscopical evidence suggests that an intermediary compound of curcumin oxidation, probably spiroepoxide, is responsible. Preincubation of curcumin in the presence of NADPH, but not glutathione disulfide (GSSG), resulted in the loss of its inhibitory ability, suggesting a reductive stabilizing effect. Similarly, preincubation of curcumin with sulfhydryl compounds fully protected the enzyme from inhibition. Degradation products were tested for their inhibitory potential, and 4-vinylguaiacol was the best inhibitor (IC50 = 12.9 µM), followed by feruloylmethane (IC50 = 122 µM), vanillin (IC50 = 127 µM), and ferulic aldehyde (IC50 = 180 µM). The acid derivatives ferulic acid (IC50 = 465 µM) and vanillic acid (IC50 = 657 µM) were poor inhibitors. On the other hand, results from docking analysis revealed a common binding site on the enzyme for all the compounds, albeit interacting with different amino acid residues. Dissociation constants obtained from the docking were in accord with the inhibitory efficiency of the curcumin degradation products.


Assuntos
Anti-Helmínticos/farmacologia , Curcumina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Taenia/enzimologia , Animais , Anti-Helmínticos/química , Sítios de Ligação , Curcumina/farmacologia , Inibidores Enzimáticos/química , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Simulação de Acoplamento Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Ligação Proteica , Taenia/efeitos dos fármacos
19.
Food Chem ; 297: 124983, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253271

RESUMO

To improve the industrial application of wheat bran insoluble dietary fiber (W-IDF), three modification methods (carboxymethylation, complex enzymatic hydrolysis, and ultrafine comminution) were compared on the basis of structural, physicochemical, functional, and antioxidant properties of W-IDF. FT-IR, DSC and SEM analysis showed that modifications contributed to alteration in morphology and arrangement of chemical bonds in W-IDF. Carboxymethylation effectively improved the water retention (WRC), water swelling (WSC), and glucose adsorption capacities (GAC); complex enzymatic hydrolysis greatly improved the oil retention (ORC), GAC, and nitrite ion adsorption capacities (NIAC). Although ultrafine comminution reduced the WRC and ORC, while positively influenced the GAC and NIAC. Moreover, total phenol content, total antioxidant capacity, DPPH radical scavenging capacity, Fe2+ chelating capacity and total reducing power were improved in modified W-IDF. Our results confirmed that carboxymethylation can improve the nutritive quality and sensory properties of W-IDF (nutritive ingredient) in food products.


Assuntos
Antioxidantes/análise , Fibras na Dieta/análise , Complexos Multienzimáticos/metabolismo , Triticum/química , Adsorção , Antioxidantes/metabolismo , Fenômenos Químicos , Fibras na Dieta/metabolismo , Glucose/metabolismo , Hidrólise , Metilação , Valor Nutritivo , Material Particulado , Silicones
20.
Mol Pharmacol ; 96(2): 212-218, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175182

RESUMO

The α7 nicotinic acetylcholine receptor (nAChR) is a potential drug target for the treatment of a number of neurologic and inflammatory disorders. Silent agonists are an emerging class of drugs that bind to the receptor but do not open the channel. Instead they shift the receptor to a desensitized state. Silent agonists may be able to target a subset of α7 nAChR-mediated signaling processes. Here we use noncanonical amino acid mutagenesis to characterize the binding to α7 by the silent agonist 1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)phenyl)furan-2-yl)methanone (NS6740). We find that, like α7 agonists, NS6740 forms a cation-π interaction with Y115 (TyrA). We also showed that NS6740 makes a novel hydrogen bond to TyrA. This interaction is necessary for the silent agonist activity of NS6740; when the hydrogen bond is blocked, silent agonist NS6740 converts to a conventional partial agonist and appreciably opens the channel in the absence of a positive allosteric modulator (EC50 150 nM). SIGNIFICANCE STATEMENT: Noncanonical amino acids were used to show that a hydrogen bond to tyrosine (Y115) is required for silent agonist activity of NS6740 at the α7 nicotinic acetylcholine receptor.


Assuntos
Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/metabolismo , Furanos/farmacologia , Complexos Multienzimáticos/metabolismo , Mutação , Receptor Nicotínico de Acetilcolina alfa7/química , Regulação Alostérica , Animais , Compostos Azabicíclicos/química , Proteínas de Bactérias/química , Furanos/química , Ligações de Hidrogênio , Complexos Multienzimáticos/química , Ligação Proteica , Ratos , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/genética
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