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1.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
2.
Chem Commun (Camb) ; 55(66): 9761-9764, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31355386

RESUMO

The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for ß-sheet formation.


Assuntos
Conformação Proteica em Folha beta , Seda/química , Animais , Cloretos/química , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Sódio/química , Solubilidade , Aranhas
3.
Yakugaku Zasshi ; 139(7): 989-992, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257257

RESUMO

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.


Assuntos
Baculoviridae/genética , Insetos/genética , Proteínas Priônicas/química , Animais , Sistema Livre de Células , Glicosaminoglicanos , Heparina , Heparitina Sulfato , Técnicas In Vitro , Ácidos Nucleicos , Proteínas Recombinantes/química
4.
Adv Mater ; 31(33): e1902462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31265196

RESUMO

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Cisteína Endopeptidases/química , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/química , Proteínas Recombinantes/química , Aminoaciltransferases/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Dissulfetos/química , Liberação Controlada de Fármacos , Humanos , Luz , Oxirredução , Peptídeos/química , Fotólise , Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Staphylococcus aureus/enzimologia
5.
Biochemistry (Mosc) ; 84(4): 390-397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228930

RESUMO

Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by heterologous expression in Escherichia coli and purified by affinity metal-chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono- and divalent cations and NADP+ as a cofactor. The kcat/Km ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C-region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyltransferase activity was detected either for a full chimeric malic enzyme or for the C-end fragment obtained as a separate protein. This C-end domain promoted activity of the malic enzyme.


Assuntos
Malato Desidrogenase/metabolismo , Methylosinus trichosporium/enzimologia , Biocatálise , Descarboxilação , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/genética , NADP/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
6.
Protein Pept Lett ; 26(5): 377-385, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237199

RESUMO

BACKGROUND: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. OBJECTIVE: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. METHODS: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. RESULTS: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively. CONCLUSION: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.


Assuntos
Aspergillus fumigatus/enzimologia , Celulose/química , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Polissacarídeos/química , Saccharum/química , Biomassa , Escherichia coli/genética , Etanol/química , Proteínas Fúngicas/genética , Hidrólise , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
7.
Biochemistry (Mosc) ; 84(6): 672-685, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238867

RESUMO

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Corpos de Inclusão/metabolismo , Porinas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Microscopia Eletrônica de Varredura , Porinas/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
8.
Phytochemistry ; 165: 112050, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31252202

RESUMO

In higher plants, asparagine-linked oligosaccharides (N-glycans) in glycoproteins carry unique carbohydrate epitopes, namely, a core α1,3-fucose and/or a ß1,2-xylose, which are common determinants responsible for the cross-reactivity of plant glycoproteins due to their strong immunogenicity. While these determinants and the relevant genes have been well characterized for herbaceous plants, information concerning whether many food plants cross-react with airborne pollens is not available. In this paper, we report on the characterization of a novel core α1,3-fucosyltransferase gene identified from Mangifera indica L., one of the major plants potentially related to food allergy. Based on sequence information of plant homologues, we amplified a candidate cDNA (MiFUT11) from pericarp tissue. An in vitro assay demonstrated that the recombinant MiFUT11 protein transfers a fucose unit onto both non-fucosylated and core α1,6-fucosylated oligosaccharides. A glycoform analysis using MALDI-TOF mass spectrometry showed that the introduction of the MiFUT11 cDNA increased the production of a core α1,3- and α1,6-fucosylated pauci-mannosidic oligosaccharide in Spodoptera Sf21 cells. Our findings suggest that MiFUT11 is a functional core α1,3-fucosyltransferase gene that is involved in the assembly of cross-reactive N-glycans in mango fruit.


Assuntos
Carboidratos/biossíntese , Frutas/química , Fucosiltransferases/metabolismo , Mangifera/enzimologia , Sequência de Aminoácidos , Carboidratos/genética , Carboidratos/imunologia , Frutas/imunologia , Frutas/metabolismo , Fucosiltransferases/química , Fucosiltransferases/genética , Mangifera/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
9.
Food Chem ; 297: 124925, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253266

RESUMO

A novel lipase gene (McLipB) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The deduced amino acid sequence of the lipase (McLipB) shared the highest identity of 46% with the Candida rugosa lipase LIP4. The extracellular lipase activity of 4304 U/mL with protein concentration of 7.7 mg/mL was achieved in a 5-L fermentor. The optimal pH and temperature of McLipB were 7.5 and 40 °C, respectively. The lipase showed high specificity towards triglycerides with short and medium chain fatty acids, and had non-position specificity. McLipB hydrolyzed butter to produce mainly butyric acid, hexanoic acid and a small amount of octanoic acid and decanoic acid. Furthermore, it degraded more than 90% dipropyl phthalate, dibutyl phthalate and dihexyl phthalate to their corresponding monoalkyl phthalates. The properties of McLipB indicate that it has great application potential for production of lipolyzed milkfat flavor and biodegradation of phthalate esters.


Assuntos
Ascomicetos/enzimologia , Lipase/metabolismo , Sequência de Aminoácidos , Técnicas de Cultura Celular por Lotes , Candida/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Lipase/genética , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , Temperatura Ambiente , Triglicerídeos/metabolismo
10.
Nat Plants ; 5(6): 589-594, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182839

RESUMO

Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development1. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs2. Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor3-5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 or brassinazole8, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Brassinosteroides/biossíntese , Sistema Enzimático do Citocromo P-450/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/antagonistas & inibidores , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Triazóis/química
11.
Food Chem ; 295: 653-661, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174809

RESUMO

Although ß-xylosidases have a wide range of applications, cold-active ß-xylosidases have been poorly studied. In this study, a cold active ß-xylosidase gene (xyl) from Bacillus pumilus TCCC 11,350 was cloned and overexpressed in Escherichia coli. The recombinant XYL (rXYL) was revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities. Purified rXYL was most active at 30 °C, demonstrating 26% and 18% of its maximum activity at 4 °C and 0 °C, respectively. Meanwhile, rXYL showed a 52% activity in 200 mM xylose, indicating a relatively strong tolerance to xylose. Moreover, rXYL exhibited a high synergistic effect (11.14-fold and 16.21-fold) with endo-xylanase to degrade beechwood xylan in both sequential and simultaneous reactions at low temperatures. As the first report on the novel cold-adapted ß-xylosidase from B. pumilus, these results suggested rXYL had attractive properties for food industrial utilizations.


Assuntos
Bacillus pumilus/enzimologia , Xilosidases/metabolismo , Sequência de Aminoácidos , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura Ambiente , Xilanos/metabolismo , Xilose/metabolismo , Xilosidases/classificação , Xilosidases/genética
12.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
13.
Nat Commun ; 10(1): 2649, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201333

RESUMO

In human and other mammalian cells, transport of L-lactate across plasma membranes is mainly catalyzed by monocarboxylate transporters (MCTs) of the SLC16 solute carrier family. MCTs play an important role in cancer metabolism and are promising targets for tumor treatment. Here, we report the crystal structures of an SLC16 family homologue with two different bound ligands at 2.54 and 2.69 Å resolution. The structures show the transporter in the pharmacologically relevant outward-open conformation. Structural information together with a detailed structure-based analysis of the transport function provide important insights into the molecular working mechanisms of ligand binding and L-lactate transport.


Assuntos
Proteínas de Bactérias/química , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Transporte de Íons/fisiologia , Ligantes , Transportadores de Ácidos Monocarboxílicos/isolamento & purificação , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/química , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Simportadores/química
14.
Nat Commun ; 10(1): 2714, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221956

RESUMO

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Proteínas de Drosophila/metabolismo , Simulação de Dinâmica Molecular , Sódio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/isolamento & purificação , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Células HEK293 , Humanos , Ligantes , Espectrometria de Massas , Conformação Proteica em alfa-Hélice/fisiologia , Domínios Proteicos/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
15.
Nat Commun ; 10(1): 2747, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227715

RESUMO

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection.


Assuntos
Proteínas de Bactérias/ultraestrutura , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/patogenicidade , Corpos de Inclusão/microbiologia , Fusão de Membrana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HeLa , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas SNARE/química
16.
Chem Biol Interact ; 309: 108698, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176713

RESUMO

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.


Assuntos
Acetilcolinesterase/química , Cristalografia por Raios X , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Aminacrina/química , Aminacrina/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Dimerização , Humanos , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente
17.
Chem Biol Interact ; 309: 108712, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31201777

RESUMO

The recent intentional use of nerve agents and pesticides in Europe and Afghanistan highlights the need for an effective countermeasure against organophosphates (OP) toxins. The most developed pretreatment candidate to date is plasma (native) human butyrylcholinesterase (HuBChE), which is limited in availability and because of its 1:1 stoichiometry with OPs, a large dose will present challenges when delivered parenterally both in terms of pharmacokinetics and manageability in the field. A tetrameric recombinant (r) form of human BChE produced in CHO-K1 cells with similar structure, in vivo stability and antidotal efficacy as the native form, has been developed to deliver rHuBChE as an aerosol (aer) to form a pulmonary bioshield capable of neutralizing inhaled OPs in situ and prevent AChE inhibition in the blood and in the brain; the latter associated with the symptoms of OP toxicity. Previous proof-of-concept macaque studies demonstrated that delivery of 9 mg/kg using a microsprayer inserted down the trachea, resulted in protection against an inhaled dose of 15ug/kg of aer-paraoxon (aer-Px) given 72 h later. In the present studies, pulmonary delivery of rHuBChE in macaques was achieved using Aerogen vibrating mesh nebulizers, similar to that used for human self-administration. The promising findings indicate that despite the poor lung deposition observed in macaques using nebulizers (13-20%), protective levels of RBC-AChE were still present in the blood even when exposure aer-Px (55 µg/kg) was delayed for five days. This long term retention of 5 mg/kg rHuBChE deposited in the lung bodes well for the use of an aer-rHuBChE pretreatment in humans where a user-friendly customized nebulizer with increased lung deposition up to 50% will provide even longer protection at a lower dose.


Assuntos
Aerossóis/química , Butirilcolinesterase/química , Paraoxon/química , Animais , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Pulmão/metabolismo , Macaca , Masculino , Nebulizadores e Vaporizadores , Paraoxon/toxicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química
18.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
19.
Chem Biol Interact ; 307: 179-185, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063765

RESUMO

The effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity.


Assuntos
Carnitina Aciltransferases/metabolismo , Mitocôndrias/metabolismo , Polifenóis/metabolismo , Prunus avium/química , Sítios de Ligação , Carnitina Aciltransferases/química , Carnitina Aciltransferases/genética , Humanos , Peróxido de Hidrogênio/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Polifenóis/análise , Estrutura Terciária de Proteína , Prunus avium/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray
20.
Rev Sci Instrum ; 90(4): 043110, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31043005

RESUMO

Raman spectroscopy has proven to be an effective tool for molecular analysis in different applications. In clinical diagnostics, its application has enabled nondestructive investigation of biological tissues and liquids. The human perilymph, for example, is an inner ear liquid, essential for the hearing sensation. The composition of this liquid is correlated with pathophysiological parameters and was analyzed by extraction and mass spectrometry so far. In this work, we present a fiber optic probe setup for the Raman spectroscopic sampling of inner ear proteins in solution. Multivariate data analysis is applied for the discrimination of individual proteins (heat shock proteins) linked to a specific type of hearing impairment. This proof-of-principle is a first step toward a system for sensitive and continuous in vivo perilymph investigation in the future.


Assuntos
Tecnologia de Fibra Óptica/métodos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Perilinfa/metabolismo , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Algoritmos , Aspirina/química , Calibragem , Desenho de Equipamento , Escherichia coli , Tecnologia de Fibra Óptica/instrumentação , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Humanos , Análise dos Mínimos Quadrados , Análise Multivariada , Perilinfa/química , Análise de Componente Principal , Prolina/química , Estudo de Prova de Conceito , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Regressão , Processamento de Sinais Assistido por Computador
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