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1.
Phys Chem Chem Phys ; 21(37): 20727-20742, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31509121

RESUMO

The left-handed polyproline II (PPII) type helical structures are thought to play a very important role in many essential biological processes, particularly in recognition mechanisms. However, reliable characterisation of PPII conformation in solution can be experimentally challenging. Computational simulation of these structures offers an attractive alternative, but the accuracy of the results is dependent on the accuracy of the force field employed. In this report, we present the results of simulation of the structural and dynamical properties of a proline-rich viral fusion peptide for which a solution NMR study reported a substantial stretch of PPII conformation in the central region. The suggested mode of action of the p15 fusion peptide depended on the exposure of the flanking N-terminal hydrophobic residues to solvent thereby facilitating their interaction with the target membrane. Our simulations with a set of four force field and water model combinations consisting of (AMBER ff99SB*-ILDNP + TIP3P), (OPLS-AA + SPC/E), (AMBER ff03ws + TIP4P/2005 water with scaled protein-water interactions) and (CHARMM36m + TIP3P) showed a general agreement with the NMR results for all the four force field and water model combinations. The central region encompassing positions 9-15 showed a large PPII propensity, reduced flexibility and lower conformational entropy. The PPII conformations were stable and satisfied the Burgi-Dunitz criteria without the participation of any significant water bridging interaction. However, comparison of the experimentally observed chemical shifts with the distribution of shifts predicted from the simulated ensembles showed a much better agreement for the CHARMM36m + TIP3P and AMBER ff03ws + TIP4P/2005 combinations. The models based on these two force fields also generated conformations which were in much better agreement with the NMR model than the much more compact structures predicted by the AMBER ff99SB*-ILDNP and OPLS-AA force fields and predicted a substantially larger solvent accessible surface area in accordance with the suggested mechanism of action of the peptide.


Assuntos
Modelos Moleculares , Prolina/química , Proteínas Recombinantes de Fusão/química , Simulação por Computador , Espectroscopia de Ressonância Magnética , Conformação Molecular , Água/química
2.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
3.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 578-591, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205020

RESUMO

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (ßFXIIaHis) with yields of ∼1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the ßFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.


Assuntos
Fator XIIa , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/química , Clorometilcetonas de Aminoácidos/química , Animais , Sítios de Ligação , Linhagem Celular , Cristalização , Cristalografia por Raios X/métodos , Drosophila melanogaster , Fator XIIa/química , Fator XIIa/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato
4.
Int J Oncol ; 55(1): 309-319, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180549

RESUMO

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Exotoxinas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Albuminas/administração & dosagem , Albuminas/química , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Linhagem Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Camundongos , Neoplasias/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética
5.
Anal Bioanal Chem ; 411(19): 4987-4998, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31254054

RESUMO

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Orthomyxoviridae/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Virais/metabolismo , Amidas/metabolismo , Células HEK293 , Humanos , Limite de Detecção , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/classificação
6.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 324-331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045561

RESUMO

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.


Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Halogenados/química , Hidrolases/química , Psychrobacter/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Psychrobacter/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
7.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 348-358, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045564

RESUMO

Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Šresolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Shewanella/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shewanella/metabolismo , Especificidade por Substrato
8.
Microb Pathog ; 132: 275-281, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078709

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)2A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.


Assuntos
Antígenos de Protozoários/imunologia , Imunogenicidade da Vacina/imunologia , Vacinas Protozoárias/imunologia , Proteínas Recombinantes de Fusão/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Biologia Computacional , Simulação por Computador , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Expressão Gênica , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Toxoplasmose/imunologia , Toxoplasmose/prevenção & controle
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 5-11, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102351

RESUMO

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-ß-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -ß-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS: Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.


Assuntos
Pirofosfatases/química , Canais de Cátion TRPM/química , Canais de Cátion TRPM/isolamento & purificação , Escherichia coli/genética , Glucosídeos/química , Humanos , Domínios Proteicos , Estabilidade Proteica , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/metabolismo
10.
Drug Des Devel Ther ; 13: 1059-1068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040643

RESUMO

Introduction: In this study, we report on the development of an effective delivery system for siRNAs; a novel cell-penetrating peptide (CPP), T9(dR), obtained from transportan (TP), was used for in vivo and in vitro testing. Methods: In this study, toxicity of T9(dR) and TP and efficient delivery of siRNA were tested in 293T, MDCK, RAW, and A549 cells. Furthermore, T9(dR)- and TP-delivered siRNAs against nucleoprotein (NP) gene segment of influenza virus (siNP) were studied in both cell lines and mice. Results: Gel retardation showed that T9(dR) effectively condensed siRNA into nanoparticles sized between 350 and 550 nm when the mole ratio of T9(dR) to siRNA was ≥4:1. In vitro studies demonstrated that T9(dR) successfully delivered siRNA with low cellular toxicity into several cell lines. It was also observed that T9(dR)-delivered siRNAs inhibited replication of influenza virus more efficiently as compared to that delivered by TP into the MDCK and A549 cells. It was also noticed that when given a combined tail vein injection of siNP and T9(dR) or TP, all mice in the 50 nmol siNP group infected with PR8 influenza virus survived and showed weight recovery at 2 weeks post-infection. Conclusion: This study indicates that T9(dR) is a promising siRNA delivery tool with potential application for nucleotide drug delivery.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Galanina/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/crescimento & desenvolvimento , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Replicação Viral/efeitos dos fármacos , Venenos de Vespas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Galanina/química , Células Madin Darby de Rim Canino , RNA Interferente Pequeno/química , Proteínas Recombinantes de Fusão/química , Venenos de Vespas/química
11.
PLoS Genet ; 15(4): e1007786, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946740

RESUMO

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Trissacarídeos/metabolismo , Sequência de Aminoácidos , Cerveja/microbiologia , Proteínas de Transporte/química , Evolução Molecular Direcionada , Fermentação , Proteínas Fúngicas/química , Conversão Gênica , Genes Fúngicos , Hibridização Genética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/química , Saccharomyces/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Simportadores/química , Simportadores/genética , Simportadores/metabolismo
12.
PLoS Genet ; 15(4): e1007853, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946741

RESUMO

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Trissacarídeos/metabolismo , Cerveja/microbiologia , Proteínas de Transporte/química , Evolução Molecular Direcionada , Fermentação , Proteínas Fúngicas/química , Genes Fúngicos , Hibridização Genética , Maltose/metabolismo , Modelos Moleculares , Mutagênese , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Recombinação Genética , Saccharomyces/crescimento & desenvolvimento , Sequenciamento Completo do Genoma
14.
Int J Mol Sci ; 20(6)2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934614

RESUMO

Carbonic anhydrases (CAs) represent a group of enzymes that catalyse important reactions of carbon dioxide hydration and dehydration, a reaction crucial to many biological processes and environmental biotechnology. In this study we successfully constructed a thermostable fusion enzyme composed of the Sulfurihydrogenibium azorense carbonic anhydrase (Saz_CA), the fastest CA discovered to date, and the chitin binding domain (ChBD) of chitinase from Bacillus circulans. Introduction of ChBD to the Saz_CA had no major impact on the effect of ions or inhibitors on the enzymatic activity. The fusion protein exhibited no negative effects up to 60 °C, whilst the fusion partner appears to protect the enzyme from negative effects of magnesium. The prepared biocatalyst appears to be thermally activated at 60 °C and could be partially purified with heat treatment. Immobilisation attempts on different kinds of chitin-based support results have shown that the fusion enzyme preferentially binds to a cheap, untreated chitin with a large crystallinity index over more processed forms of chitin. It suggests significant potential economic benefits for large-scale deployment of immobilised CA technologies such as CO2 utilisation or mineralisation.


Assuntos
Bactérias/enzimologia , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Quitina/metabolismo , Proteínas Imobilizadas/metabolismo , Proteínas Recombinantes de Fusão/química , Temperatura Ambiente , Água/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/isolamento & purificação , Cristalização , Estabilidade Enzimática , Íons , Peso Molecular , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo
15.
Molecules ; 24(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991754

RESUMO

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.


Assuntos
Proteínas Fúngicas , Expressão Gênica , Pichia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
16.
Enzyme Microb Technol ; 126: 69-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000166

RESUMO

The beta-propeller phytase (BPP) is an enzyme that hydrolyzes phyate to release inorganic phosphorus. The BPP produced by Pseudomonas sp. FB15 (PSphy) possesses an additional N-terminal domain that is not present in BPP produced by other Bacillus species. In this study, BPP produced by Bacillus sp. SJ-10 (SJ-10phy) was fused with the N-terminal domain of PSphy and the enzymatic properties of the resulting fusion protein (FUSJ-10phy) were investigated. FUSJ-10phy exhibited an optimal temperature that was 10 °C lower than that of the wild-type enzyme. A comparison of kinetic parameters showed that the turnover rate of FUSJ-10phy was 2.39-fold higher than that of SJ-10phy, representing a 1.79-fold increase in catalytic efficiency. In addition, BPP produced by Bacillus sp. SJ-48 has relatively low sequence similarity with SJ-10phy, was fused with N-terminal domain (FUSJ-48phy). FUSJ-48phy also increased catalytic efficiency and changed the optimal temperature. These results indicate that, when fused to other BPPs, the N-terminal domain of PSphy increases catalytic efficiency and enzyme activity at lower temperatures.


Assuntos
6-Fitase/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Pseudomonas/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Temperatura Ambiente , 6-Fitase/química , 6-Fitase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência
17.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940700

RESUMO

The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Salmonella typhimurium/fisiologia , Movimento (Física) , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Supressão Genética
18.
RNA ; 25(6): 685-701, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30910870

RESUMO

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , RNA Helicases DEAD-box/química , Biogênese de Organelas , RNA Helicases/química , RNA/química , Ribonucleoproteínas Nucleolares Pequenas/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
19.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832342

RESUMO

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.


Assuntos
Radioisótopos de Gálio/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-3/metabolismo , Acetatos/química , Animais , Linhagem Celular Tumoral , Quelantes/química , Compostos Heterocíclicos com 1 Anel/química , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Compostos Radiofarmacêuticos/química , Proteínas Recombinantes de Fusão/química , Distribuição Tecidual
20.
Mol Vis ; 25: 165-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820152

RESUMO

Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.


Assuntos
Sequência de Bases , Proteínas do Olho/genética , Proteínas Recombinantes de Fusão/genética , Retina/metabolismo , Deleção de Sequência , Ativação Transcricional , Proteínas de Xenopus/genética , Motivos de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis
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