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1.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366154

RESUMO

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.


Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química
2.
Molecules ; 24(17)2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466351

RESUMO

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.


Assuntos
DNA/metabolismo , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Substituição de Aminoácidos , Arginina/genética , Domínio Catalítico , Dano ao DNA , Histidina/genética , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fenilalanina/genética , Ligação Proteica , Uracila-DNA Glicosidase/genética
3.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31321399

RESUMO

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Assuntos
Hidrolases/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálise , Cobre/química , Histidina/química , Histidina/genética , Hidrolases/genética , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/química , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Treonina/química , Zinco/química
4.
Nat Commun ; 10(1): 3067, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296851

RESUMO

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Staphylococcus aureus/metabolismo , Zinco/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cátions Bivalentes/metabolismo , Histidina/genética , Histidina Quinase/química , Histidina Quinase/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Regulon/genética , Staphylococcus aureus/genética , Tirosina/genética
5.
Biochemistry (Mosc) ; 84(5): 520-528, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234766

RESUMO

In our recent X-ray study, we demonstrated that substitution of the natural leucine residue M196 with histidine in the reaction center (RC) from Rhodobacter (Rba.) sphaeroides leads to formation of a close contact between the genetically introduced histidine and the primary electron donor P (bacteriochlorophylls (BChls) PA and PB dimer) creating a novel pigment-protein interaction that is not observed in native RCs. In the present work, the possible nature of this novel interaction and its effects on the electronic properties of P and the photochemical charge separation in isolated mutant RCs L(M196)H are investigated at room temperature using steady-state absorption spectroscopy, light-induced difference FTIR spectroscopy, and femtosecond transient absorption spectroscopy. The results are compared with the data obtained for the RCs from Rba. sphaeroides pseudo-wild type strain. It is shown that the L(M196)H mutation results in a decrease in intensity and broadening of the long-wavelength Qy absorption band of P at ~865 nm. Due to the mutation, there is also weakening of the electronic coupling between BChls in the radical cation P+ and increase in the positive charge localization on the PA molecule. Despite the significant perturbations of the electronic structure of P, the mutant RCs retain high electron transfer rates and quantum yield of the P+QA- state (QA is the primary quinone acceptor), which is close to the one observed in the native RCs. Comparison of our results with the literature data suggests that the imidazole group of histidine M196 forms a π-hydrogen bond with the π-electron system of the PB molecule in the P dimer. It is likely that the specific (T-shaped) spatial organization of the π-hydrogen interaction and its potential heterogeneity in relation to the bonding energy is, at least partially, the reason that this type of interaction between the protein and the pigment and quinone cofactors is not realized in the native RCs.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Leucina/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Transporte de Elétrons , Histidina/genética , Cinética , Leucina/genética , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier
6.
Analyst ; 144(12): 3773-3781, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089613

RESUMO

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.


Assuntos
Bioensaio/métodos , Proteínas Luminescentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Cromatografia de Afinidade/métodos , Complexos de Coordenação/química , Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Humanos , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/química , Platina/química , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética
7.
Prep Biochem Biotechnol ; 49(5): 529-534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31030612

RESUMO

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD+ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD+ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.


Assuntos
Chaetomium/enzimologia , Formiato Desidrogenases/química , Proteínas Fúngicas/química , Histidina/química , Proteínas Recombinantes/química , Catálise , Ensaios Enzimáticos , Formiato Desidrogenases/genética , Formiato Desidrogenases/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Histidina/genética , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade
8.
PLoS One ; 14(3): e0214319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913245

RESUMO

Porcine gamma interferon is a cytokine produced by activated T cells and NK cells with broad-spectrum antiviral activity and immunomodulatory function. However, pIFN-γ is a secretory protein that has a short half-life in organisms and is easily inactivated, making it difficult to apply widely in clinics. Therefore, we tried to optimize the expression of pIFN-γ in Pichia pastoris to obtain a large amount of highly active, easily purified pIFN-γ protein in vitro. Through C-terminal sequence analysis, we found a signal sequence (EKREAEAE) that was easily enzymolysed by a signal peptide enzyme, resulting in degradation and inactivation of the pIFN-γ protein. In this study, we optimized the pIFN-γ gene recombination sequence and mutated the 3' end of the pIFN-γ gene, resulting in a higher expression level and stronger biological activity, as well as a significant upregulation in the expression of the interferon-stimulated genes Mx1 and OAS1 in IPEC-J2 jejunal epithelial cells. Our data also showed that the fermentation process could significantly improve productivity. A recombinant Pichia pastoris strain with the optimized pIFN-γ gene could obtain a high yield of pIFN-γ protein, up to 9536 mg/L, after staged incubation for 0-24 h at 28°C, pH 6.0, and 50% dissolved oxygen (DO), followed by incubation for 24-72 h at 25°C, pH 6.0 and 30% DO. These data demonstrated, for the first time, that the expression level of pIFN-γ in Pichia pastoris was improved significantly by gene optimization with 3' end mutation and a fermentation process that maintained good biological activity, which is beneficial to the application of pIFN-γ in animal husbandry.


Assuntos
Interferon gama/metabolismo , Pichia/metabolismo , Sinais Direcionadores de Proteínas/genética , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Técnicas de Cultura Celular por Lotes , Linhagem Celular , Histidina/genética , Histidina/metabolismo , Interferon gama/genética , Mutação , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Suínos
9.
Nat Chem ; 11(5): 434-441, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30778140

RESUMO

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein-protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres-including a penta-His-coordinated non-haem Fe site-and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.


Assuntos
Grupo dos Citocromos b/metabolismo , Proteínas de Escherichia coli/metabolismo , Metaloproteínas/metabolismo , Metais Pesados/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Cisteína/química , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Dissulfetos/química , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina/química , Histidina/genética , Metaloproteínas/genética , Mutação , Óxido Nítrico/metabolismo , Ligação Proteica
10.
Anal Biochem ; 570: 62-64, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30735667

RESUMO

We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the His-tag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies.


Assuntos
Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fosfatase Alcalina/metabolismo , Histidina/genética , Histidina/metabolismo , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosfopeptídeos/análise , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo
11.
Prep Biochem Biotechnol ; 49(2): 167-175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689537

RESUMO

To enhance recovery yield of carboxymethylcellulase (CMCase), E. coli BL21/A-53 producing the histidine-tagged CMCase was constructed in this study. The recovery yield of the histidine-tagged CMCase using the His-tag affinity chromatography was 39.8%. The predicted molecular weight of the histidine-tagged CMCase was determined as 56,260 Da. Its Km and Vmax were 9.3 g l-1 and 76.3 g l-1·min-1, respectively. The histidine-tagged CMCase hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, xylan, but there was no detectable activity on cellobiose, p-Nitrophenyl-ß-D-glucopyranoside (pNPG). The optimal temperature and pH for the enzymatic reaction of the histidine-tagged CMCase was 50 °C and 5.0. The histidine-tagged CMCase was enhanced by CoCl2 until the concentration of 100 mM, but inhibited by EDTA, HgCl2, MnCl2, NiCl2, and RbCl2. The characteristics of the histidine-tagged CMCase produced by E. coli BL21/A-53 were compared with those of CMCase without the histidine-tag of Bacillus subtilis subsp. subtilis A-53. The little changed characteristics of the histidine-tagged CMCase compared to the CMCase without a His-tag seemed to be the conformational change in the structure due to a His-tag.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Celulase/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Histidina/genética , Oligopeptídeos/genética , Sequência de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Celulase/química , Celulase/metabolismo , Cromatografia de Afinidade/métodos , Estabilidade Enzimática , Escherichia coli/química , Escherichia coli/metabolismo , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Muscle Nerve ; 59(1): 129-133, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30230566

RESUMO

INTRODUCTION: Mutations in the TRPV4 gene are associated with neuromuscular disorders and skeletal dysplasias, which present a phenotypic overlap. METHODS: Next-generation sequencing and Sanger sequencing were used to analyze the TRPV4 gene. RESULTS: We present 2 Polish families with TRPV4-related disorder harboring the same p.Arg269His mutation. The disease phenotypic expression was extremely variable (from mild scapular winging to severe hypotonia, global weakness, inability to walk unaided, congenital contractures, scoliosis, and respiratory insufficiency), but did not suggest anticipation. The 2 most severely affected patients showed congenital distal contractures of the upper limbs and involvement of cranial nerves (manifesting as facial asymmetry and strabismus). The disease course seemed to be stable, although in later stages it caused respiratory insufficiency and progression of physical disability. DISCUSSION: The phenotypic variability observed in p.Arg269His carriers suggests that an additional modifier or a more complex pathogenic mechanism exists. Muscle Nerve 59:129-133, 2019.


Assuntos
Arginina/genética , Histidina/genética , Atrofia Muscular Espinal/genética , Mutação/genética , Canais de Cátion TRPV/genética , Adulto , Pré-Escolar , Creatina Quinase/sangue , Saúde da Família , Feminino , Heterozigoto , Humanos , Masculino , Atrofia Muscular Espinal/sangue , Atrofia Muscular Espinal/patologia , Transaminases/sangue
13.
Int J Biol Macromol ; 124: 810-818, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500497

RESUMO

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.


Assuntos
Bacteriófagos/química , Endopeptidases/química , Histidina/química , Metaloproteínas/química , Oligopeptídeos/química , Proteínas Virais/química , Zinco/química , Bacteriófagos/enzimologia , Cálcio/química , Cálcio/metabolismo , Domínio Catalítico , Cátions Bivalentes , Cromatografia de Afinidade , Clonagem Molecular , Diálise/métodos , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Imidazóis/química , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Metaloproteínas/metabolismo , Níquel/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo , Zinco/metabolismo
14.
Protein Sci ; 28(1): 100-110, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30056630

RESUMO

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y revealed a preference for H2 O2 as an oxidant with second order rate constants for H2 O2 and tBuOOH around 2.5 × 107 M-1 s-1 and 3 × 106 M-1 s-1 , respectively. Differences between the oxidation kinetics of PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.


Assuntos
Substituição de Aminoácidos , Histidina/química , Peroxirredoxina VI/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Tirosina/química , Domínio Catalítico , Ativação Enzimática/genética , Mutação com Ganho de Função , Histidina/genética , Peróxido de Hidrogênio/química , Cinética , Oxirredução , Peroxirredoxina VI/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Tirosina/genética
15.
Molecules ; 23(12)2018 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-30544954

RESUMO

The protein absent in melanoma 1 (AIM1) is a member of the ßγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to ß-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the ß-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and ß-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and ß-actin; we also obtain the crystal of the first two ßγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the ßγ-crystallin domain repeats that bound to ß-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.


Assuntos
Actinas/metabolismo , Cristalinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/isolamento & purificação , Motivos de Aminoácidos , Clonagem Molecular , Cristalinas/genética , Cristalinas/isolamento & purificação , Escherichia coli/genética , Histidina/genética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Soluções
16.
J Microbiol Methods ; 155: 49-54, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30445111

RESUMO

Typical methods for elucidating the function of a particular gene involve comparative phenotypic analysis of the wild-type strain and a strain in which the gene of interest has been disrupted. We previously described a simple method for the generation of a gene-disrupted strain in Streptococcus mutans by replacing the gene of interest with an antibiotic resistance marker gene. It is also crucial that the function lost following the gene disruption is restored by exogenous addition of the gene product, but purification of this product can be difficult and involve a complex series of steps. In this study, we describe a simple method for the purification of gene products following gene disruption in S. mutans. The method involves the expression of an additional polyhistidine tag at the C-terminus of the gene product. The target protein can be simply purified by immobilized metal affinity chromatography and applied to a restoration assay. This method utilizes the genomes of both the wild-type strain and the gene-disrupted strain as PCR templates to generate the DNA construct. Therefore, generation of the gene-disrupted strain is a prerequisite for the present procedure. The combination of gene disruption and gene product purification results in an efficient method for the analysis of gene function that could be further adapted to various other bacterial species.


Assuntos
Genes Reporter/genética , Histidina/genética , Histidina/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Streptococcus mutans/genética , Adesinas Bacterianas , Biofilmes/crescimento & desenvolvimento , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Marcadores Genéticos , Recombinação Homóloga/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/genética
17.
Acta Neuropathol Commun ; 6(1): 122, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30409191

RESUMO

Mutations in ubiquilin2 (UBQLN2) have been linked to abnormal protein aggregation in amyotrophic lateral sclerosis (ALS). The mechanisms underlying UBQLN2-related neurodegenerative diseases remain unclear. Using a tetracycline-regulated gene expression system, the ALS-linked UBQLN2P497H mutant was selectively expressed in either the spinal motor neurons or astrocytes in rats. We found that selectively expressing mutant UBQLN2P497H in the spinal motor neurons caused several core features of ALS, including the progressive degeneration of motor neurons, the denervation atrophy of skeletal muscles, and the abnormal protein accumulation. Furthermore, mutant UBQLN2P497H accumulation was associated with an age-dependent decrease in several core autophagy-related proteins. ALS-like phenotypes were not observed when mutant UBQLN2P497H was overexpressed in the astrocytes, however, even though the expression of the mutant UBQLN2P497H protein was higher in these rats. Our results suggest that selectively expressing mutant UBQLN2P497H in motor neurons is sufficient to trigger the development of ALS in rats. Our results further indicate that the compromised autophagy-lysosomal pathway plays a critical role in the pathogenesis of UBQLN2-related neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Autofagia/genética , Neurônios Motores/metabolismo , Mutação/genética , Ubiquitinas/genética , Administração Oral , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Colina O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Regulação da Expressão Gênica/genética , Histidina/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Transtornos Motores/etiologia , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Prolina/genética , Desempenho Psicomotor/fisiologia , Ratos , Ratos Transgênicos , Medula Espinal/patologia
18.
J Alzheimers Dis ; 66(2): 775-787, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30320594

RESUMO

BACKGROUND: The amyloid AV-45 (florbetapir) positron emission tomography (PET) has been used in the study of the familial Alzheimer's disease (FAD) with the D678H amyloid precursor protein (APP) mutation. In addition, the progress of the disease remains unknown. OBJECTIVE: We aim to investigate the progression rate of amyloid accumulation in FAD patients with this mutation by neuroimages analysis. METHODS: The clinical course, changes in cognitive function, brain magnetic resonance imaging (MRI) and 18F-AV-45 PET scan were investigated in FAD patients and sporadic AD (sAD) patients. We compared the amyloid deposition pattern in serial brain 18F-AV-45 PET scan among the FAD, familial mild cognitive impairment (FMCI), and sMCI and sAD patients. RESULTS: Seven familial patients received a follow-up survey. The follow up duration for brain AV-45 PET was from 1.54 to 3.61 years. In 4 FMCI patients, an increased regional SUVR was noted, and the annual change rates were increased from 1.03% to 18.82%. However, a decreased regional SUVR was noted in 3 FAD patients and the annual change rates were from -2.62% to -16.03%. As compared with the sAD and sMCI patients, the annual change rate is statistically significant in FAD and FMCI patients respectively. CONCLUSIONS: The data indicate a biphasic course with an initial increase and then a decrease of SUVR in brain amyloid PET scan in familial APP mutation patients. The data also reveal that the novel Taiwan APP (D678H) mutation has a more amyloid burden than the sAD patients, particularly in an MCI stage.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Disfunção Cognitiva/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico por imagem , Compostos de Anilina/farmacocinética , Ácido Aspártico/genética , Encéfalo/diagnóstico por imagem , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/patologia , Progressão da Doença , Etilenoglicóis/farmacocinética , Saúde da Família , Feminino , Seguimentos , Histidina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Taiwan
19.
PLoS One ; 13(10): e0205870, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335802

RESUMO

In eukaryotes, the modification of an invariant histidine (His-699 in yeast) residue in translation elongation factor 2 (EF2) with diphthamide involves a conserved pathway encoded by the DPH1-DPH7 gene network. Diphthamide is the target for diphtheria toxin and related lethal ADP ribosylases, which collectively kill cells by inactivating the essential translocase function of EF2 during mRNA translation and protein biosynthesis. Although this notion emphasizes the pathological importance of diphthamide, precisely why cells including our own require EF2 to carry it, is unclear. Mining the synthetic genetic array (SGA) landscape from the budding yeast Saccharomyces cerevisiae has revealed negative interactions between EF2 (EFT1-EFT2) and diphthamide (DPH1-DPH7) gene deletions. In line with these correlations, we confirm in here that loss of diphthamide modification (dphΔ) on EF2 combined with EF2 undersupply (eft2Δ) causes synthetic growth phenotypes in the composite mutant (dphΔ eft2Δ). These reflect negative interference with cell performance under standard as well as thermal and/or chemical stress conditions, cell growth rates and doubling times, competitive fitness, cell viability in the presence of TOR inhibitors (rapamycin, caffeine) and translation indicator drugs (hygromycin, anisomycin). Together with significantly suppressed tolerance towards EF2 inhibition by cytotoxic DPH5 overexpression and increased ribosomal -1 frame-shift errors in mutants lacking modifiable pools of EF2 (dphΔ, dphΔ eft2Δ), our data indicate that diphthamide is important for the fidelity of the EF2 translocation function during mRNA translation.


Assuntos
Regulação Fúngica da Expressão Gênica , Histidina/análogos & derivados , Histidina/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Anisomicina/farmacologia , Cafeína/farmacologia , Divisão Celular/efeitos dos fármacos , Cinamatos/farmacologia , Toxina Diftérica/toxicidade , Deleção de Genes , Histidina/genética , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Metiltransferases/genética , Metiltransferases/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 2 de Elongação de Peptídeos/deficiência , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia
20.
Nature ; 562(7726): 286-290, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30283133

RESUMO

Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life1. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling2,3. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively4-10. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid in Gram-positive bacteria11-16, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure-function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Bicamadas Lipídicas/metabolismo , Aciltransferases/genética , Sequência de Aminoácidos , Animais , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Parede Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Histidina/genética , Histidina/metabolismo , Bicamadas Lipídicas/química , Lipopolissacarídeos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Relação Estrutura-Atividade , Ácidos Teicoicos/metabolismo
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