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1.
Chem Commun (Camb) ; 55(66): 9869-9872, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31364617

RESUMO

In this work, we designed and synthesized an aggregation-induced emission (AIE)-active tetraphenylethene-decorated pseudo-trisialic acid (TPE3S) and validated its high affinity for Siglecs using microscale thermophoresis techniques. TPE3S was a unique binding-on fluorescent trivalent sialocluster which was successfully utilized for the visualization of Siglecs expressed on the surface of mammalian cells.


Assuntos
Metabolismo dos Carboidratos , Lectinas/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Células PC12 , Ligação Proteica , Ratos
2.
Cell Physiol Biochem ; 53(2): 366-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385665

RESUMO

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.


Assuntos
Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Carioferinas/metabolismo , Fosforilação , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo
3.
Phys Chem Chem Phys ; 21(32): 17821-17835, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31373340

RESUMO

The rise of New Delhi metallo-beta-lactamase-1 (NDM-1) producers is a major public health concern due to carbapenem resistance. Infections caused by carbapenem-resistant enterobacteria (CRE) are classified as a serious problem. To understand the structure and function of NDM-1, an amino acid replacement approach is considered as one of the methods to get structural insight. Therefore, we have generated novel mutations (N193A, S217A, G219A and T262A) near active sites and an omega-like loop to study the role of conserved residues of NDM-1. The minimum inhibitory concentrations (MICs) of ampicillin, imipenem, meropenem, cefotaxime, cefoxitin and ceftazidime for all mutants were found to be reduced 2 to 6 fold, compared to a wild type NDM-1 producing strain. The Km values increased while Kcat and Kcat/Km values were decreased compared to wild type. The affinity as well as the catalysis properties of these mutants were reduced considerably for imipenem, meropenem, cefotaxime, cefoxitin, and ceftazidimem compared to wild type, hence the catalytic efficiencies (Kcat/Km) of all mutant enzymes were reduced owing to the poor affinity of the enzyme. The IC50 values of these mutants with respect to each drug were reduced compared to wild type NDM-1. MD simulations and docking results from the mutant protein models, along with the wild type example, showed stable and consistent RMSD, RMSF and Rg behavior. The α-helix content values of all mutant proteins were reduced by 13%, 6%, 14% and 9% compared to NDM-1. Hence, this study revealed the impact role of active sites near residues on the enzyme catalytic activity of NDM-1.


Assuntos
Antibacterianos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , beta-Lactamases/química , Antibacterianos/farmacologia , Biocatálise , Domínio Catalítico , Farmacorresistência Bacteriana , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
Acta Crystallogr C Struct Chem ; 75(Pt 8): 1091-1101, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31380792

RESUMO

A new set of differently hydrated barium and strontium squarates, namely poly[[triaqua(µ-1,2-dioxocyclobut-3-ene-1,2-diolato)barium] monohydrate], {[Ba(C4O4)(H2O)3]·H2O}n (1), poly[[diaqua(µ-1,2-dioxocyclobut-3-ene-1,2-diolato)strontium] monohydrate], {[Sr(C4O4)(H2O)2]·H2O}n (2), and poly[[triaqua(µ-1,2-dioxocyclobut-3-ene-1,2-diolato)barium/strontium(0.85/0.15)] monohydrate], {[Ba0.85Sr0.15(C4O4)(H2O)3]·H2O}n (3), is reported. The study of their crystal structures indicates that all the complexes crystallize in the triclinic space group P-1. Complexes 1 and 3 have a rare combination of squarate units coordinated through monodentate O atoms to two different metal atoms and through two bidentate O atoms to three different metal atoms. Furthermore, they have three coordinated water molecules to give a coordination number of nine. The squarate ligands in complex 2 exhibit two different coordination modes: (i) monodentate O atoms coordinated to four different Sr atoms and (ii) two monodentate O atoms coordinated to two different metal atoms with the other two O atoms bidentate to four different Sr atoms. All the compounds decompose to give the respective carbonates when heated to 800 °C, as evidenced by thermogravimetry/differential thermal analysis (TG-DTA), which are clusters of nanoparticles. Complexes 1 and 3 show additional endothermic peaks at 811 and 820 °C, respectively, indicating the phase transition of BaCO3 from an orthorhombic (α-Pmcn) to a trigonal phase (ß-R3m). All three complexes have significant DNA-binding constants, ranging from 2.45 × 104 to 9.41 × 104 M-1 against EB-CT (ethidium bromide-calf thymus) DNA and protein binding constants ranging from 1.1 × 105 to 8.6 × 105 with bovine serum albumin. The in vitro cytotoxicity of the complexes is indicated by the IC50 values, which range from 128.8 to 261.3 µg ml-1. Complex 3 shows better BSA binding, antioxidant activity against the DPPH radical and cytotoxicity than complexes 1 and 2.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ciclobutanos/farmacologia , Depuradores de Radicais Livres/farmacologia , Substâncias Intercalantes/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Bário/química , Bovinos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Ciclobutanos/síntese química , Ciclobutanos/química , Ciclobutanos/metabolismo , DNA/metabolismo , Depuradores de Radicais Livres/síntese química , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/metabolismo , Humanos , Ligações de Hidrogênio , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Ligantes , Células MCF-7 , Estrutura Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Estrôncio/química , Água/química
5.
Chem Commun (Camb) ; 55(68): 10158-10161, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389420

RESUMO

We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E. coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens.


Assuntos
Celulose/química , Escherichia coli K12/isolamento & purificação , Mananas/química , Nanofibras/química , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Técnicas de Tipagem Bacteriana/instrumentação , Técnicas de Tipagem Bacteriana/métodos , Linhagem Celular Tumoral , Celulose/metabolismo , Escherichia coli K12/química , Fezes/microbiologia , Proteínas de Fímbrias/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Mananas/metabolismo , Papel , Ligação Proteica
7.
Cell Physiol Biochem ; 53(2): 429-438, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424183

RESUMO

BACKGROUND/AIMS: Chronic kidney disease-mineral bone disorder is a major complication affecting the vast majority of chronic kidney disease patients. A hallmark of the disorder is an altered parathyroid gland biology resulting in secondary hyperparathyroidism. This condition is widely treated by calcimimetics like cinacalcet which act by allosteric activation of the calcium sensing receptor. METHODS: Here, we present a linear multi-compartment model based on physiological principles such as first-pass metabolism and protein binding, which captures all relevant pharmacokinetic parameters of cinacalcet. RESULTS: Due to the linear structure of the model, simulations are numerically stable and allow fast and accurate short or long-term predictions of cinacalcet concentrations in the body. CONCLUSION: The model compartments are physiological meaningful and can be easily adjusted to various conditions like impaired hepatic clearance or different drug administration regimens. Moreover, the model can be easily adapted to specific patient groups.


Assuntos
Calcimiméticos/farmacocinética , Cinacalcete/farmacocinética , Modelos Biológicos , Calcimiméticos/sangue , Calcimiméticos/metabolismo , Cinacalcete/sangue , Cinacalcete/metabolismo , Simulação por Computador , Humanos , Hiperparatireoidismo Secundário/sangue , Hiperparatireoidismo Secundário/tratamento farmacológico , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/metabolismo , Fígado/metabolismo , Ligação Proteica , Insuficiência Renal Crônica/complicações
8.
Anticancer Res ; 39(8): 4111-4116, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366495

RESUMO

BACKGROUND/AIM: We investigated whether the expression of inositol 1, 4, 5-trisphosphate receptor-binding protein released with inositol 1, 4, 5-trisphosphate (IRBIT) in clinical gastric cancer (GC) patients could predict the therapeutic response to postoperative adjuvant chemotherapy. MATERIALS AND METHODS: Immunohistochemistry was used to investigate IRBIT expression in 115 GC patients. To clarify whether IRBIT had a relationship with the therapeutic effects of chemotherapy, we compared two groups - 62 patients treated with postoperative adjuvant chemotherapy and 53 patients treated with postoperative adjuvant chemotherapy. RESULTS: Regarding the postoperative adjuvant chemotherapy-free group, we did not find any statistically significant correlation between clinicopathological features and recurrence regardless of the expression of IRBIT. In contrast, in the group receiving postoperative adjuvant chemotherapy, a significant association was found between IRBIT expression and both overall and disease-free survival. CONCLUSION: IRBIT may be used as a useful predictive marker for chemotherapy.


Assuntos
Biomarcadores Tumorais/genética , Lectinas Tipo C/genética , Proteínas de Membrana/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Idoso , Quimioterapia Adjuvante/efeitos adversos , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
9.
Phys Chem Chem Phys ; 21(28): 15630-15638, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31268447

RESUMO

We studied the influence of globotriaosylceramide (Gb3) lipid molecules on the properties of phospholipid membranes composed of a liquid ordered (lo)/liquid disordered (ld) phase separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM)/cholesterol mixture (40/35/20, mol/mol/mol) supplemented with 5 mol% of either short acyl chain palmitoyl-Gb3 or long acyl chain lignoceryl-Gb3 using 2H solid-state NMR spectroscopy. To this end, both globotriaosylceramides were chemically synthesized featuring a perdeuterated lipid acyl chain. The solid-state 2H NMR spectra support the phase separation into a POPC-rich ld phase and a PSM/cholesterol-rich lo phase. The long chain lignoceryl-Gb3 showed a rather unusual order parameter profile of the acyl chain, which flattens out for the last ∼6 methylene segments. Such an odd chain conformation can be explained by partial chain interdigitation and/or a very fluid midplane region of the membrane. Possibly, the Gb3 molecules may thus preferentially be localized at the lo/ld phase boundary. In contrast, the short chain palmitoyl-Gb3 was well associated with the PSM/cholesterol-rich lo phase. Gb3 molecules act as membrane receptors for the Shiga toxin (STx) produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli (EHEC). The B-subunits of STx (STxB) forming a pentameric structure were produced recombinantly and incubated with the membrane mixtures leading to alterations in the lipid packing properties and lateral organization of the membranes. Typically, STxB binding led to a decrease in lipid chain order in agreement with partial immersion of protein segments into the lipid-water interface of the membrane. In the presence of STxB, Gb3 preferentially partitioned into the lo membrane phase. In particular the short acyl chain palmitoyl-Gb3 showed very similar chain order parameters to PSM. In the presence of STxB, all lipid species showed isotropic contributions to the 2H NMR powder spectra; this was most pronounced for the Gb3 molecules. Such isotropic contributions are caused by highly curved membrane structures, which have previously been detected as membrane invaginations in fluorescence microscopy. Our analysis estimated that STxB induced highly curved membrane structures with a curvature radius of less than ∼10 nm likely related to the insertion of STxB segments into the lipid-water interface of the membrane.


Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Espectroscopia de Ressonância Magnética , Toxina Shiga/química , Ligação Proteica
10.
Phys Chem Chem Phys ; 21(28): 15787-15797, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282520

RESUMO

With the aim of improving the reproducibility of capacitive immunosensors, we performed a comparative study of four different insulating/immobilization chemistries. Each chemistry targeted different areas of an interdigitated electrode including an alkyl thiol monolayer on the electrode surface, an amino silane monolayer on the gaps between electrodes, and conformal coatings via passive adsorption of the probe and a spin-coated layer of poly(methyl methacrylate) (PMMA). We analyzed the dielectric properties of these chemistries by comparing their capacitive behavior through equivalent circuit modeling and correlate the observed behavior with their surface characteristics by using atomic force microscopy and finite element modeling. We found that surface binding events occurring in the interdigitated electrode gaps play a major role in the overall change in capacitance. This was confirmed via finite element modeling showing an increased electric field intensity in the electrode gaps by 14%, compared to directly above the electrodes. Among the investigated surface chemistries, PMMA conformal coating produced a smooth surface (Rq roughness = 0.21 ± 0.02 nm) providing the most reproducible and stable capacitance change (15.6 ± 0.4%) in response to specific antigen-antibody binding.


Assuntos
Técnicas Biossensoriais/instrumentação , Eletrodos , Imunoensaio/instrumentação , Capacitância Elétrica , Microscopia de Força Atômica , Modelos Químicos , Ligação Proteica
11.
J Chem Theory Comput ; 15(8): 4602-4614, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31268700

RESUMO

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg2+/Ca2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.


Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Metaloproteínas/metabolismo , Animais , Cálcio/química , Cátions Bivalentes/química , Bases de Dados de Proteínas , Humanos , Magnésio/química , Metaloproteínas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Software , Eletricidade Estática , Termodinâmica
12.
J Agric Food Chem ; 67(32): 8919-8925, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31334658

RESUMO

Histone deacetylase (HDAC) performs important functions in plant growth and development, including fruit ripening. As a complex biological process, fruit ripening involves the histone acetylation modification of ripening-associated genes. Histone deacetylase genes (HDACs) have been well studied in Arabidopsis and rice, but the biological functions of HDACs in papaya are poorly understood. In the present work, three CpHDACs, belonging to the RPD3/HDA1 subfamily, were identified from papaya and named as CpHDA1, CpHDA2, and CpHDA3. CpHDA1 and CpHDA2 were induced by propylene, while CpHDA3 was propylene-repressed. Moreover, CpHDA3 protein could physically interact with CpERF9 and enhance the transcriptional repression activities of CpERF9 to downstream genes CpPME1, CpPME2 and CpPG5. Histone acetylation levels of CpPME1 and CpPG5 were increased during fruit ripening. Taken together, these results suggested that CpERF9 recruits CpHDA3 to form a histone deacetylase repressor complex to mediate pectin methylesterase and polygalacturonase genes expression during papaya fruit ripening and softening.


Assuntos
Hidrolases de Éster Carboxílico/genética , Carica/metabolismo , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/metabolismo , Proteínas de Plantas/metabolismo , Poligalacturonase/genética , Fatores de Transcrição/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Carica/genética , Carica/crescimento & desenvolvimento , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Proteínas de Plantas/genética , Poligalacturonase/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética
13.
J Agric Food Chem ; 67(32): 8746-8755, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31322881

RESUMO

The underlying mechanisms of the higher photosynthetic efficiency of cultivated cassava relative to its wild species are poorly understood. In the present study, proteins in leaves and chloroplasts were analyzed to compare the differences among the cultivar SC205, its wild ancestor W14, and the related species Glaziovii. The functions of differential proteins are associated with 10 ontology groups including photosynthesis, carbohydrate and energy metabolism, as well as potential signal pathway. The protein-protein networks among 41 differential proteins showed that PGK1 is a hub protein and protein cross-interactions affected the differentiation of photosynthetic rate. Anatomy patterns and PEPC detection suggested that SC205 has more C4 photosynthesis characteristics than Glaziovii and W14. Finally, a mechanism model of the efficient photosynthesis was proposed based on the remarkable variations in photosynthetic parameters and protein functions in the domestic cultivars.


Assuntos
Manihot/metabolismo , Fotossíntese , Cloroplastos/metabolismo , Manihot/classificação , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas
14.
J Agric Food Chem ; 67(32): 8896-8904, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339308

RESUMO

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/genética , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos
15.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
16.
Chem Commun (Camb) ; 55(61): 8975-8978, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290492
17.
Vet Immunol Immunopathol ; 213: 109889, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31307671

RESUMO

Blocking immunoglobulin G (IgG) binding receptors on leukocytes is an established and highly recommended preventive procedure for immunological assays. Failing to prevent such nonspecific binding can lead to erroneous results. Several studies testing different blocking reagents have been performed in murine or human cells, however, there are no specific studies on bovine cells. Our study aimed to investigate the efficiency of blocking reagents to inhibit the nonspecific binding of mouse monoclonal antibodies (mAbs) to bovine peripheral blood cells. We observed nonspecific interactions of IgG2a and IgG2b negative isotypes with bovine leukocytes, but not IgG1. We found that these nonspecific bindings could be eliminated by blocking with purified mouse IgG, whereas little or no blocking effect was observed when bovine serum or Mouse Seroblock FcR were applied. Moreover, in the absence of an efficient blocking reagent, the percentage of CD335 positive cells was significantly higher than in the group previously blocked with mouse IgG. Based on these results, and due to the lack of specific commercial blocking reagents for bovine cells, our recommendation is to use purified mouse IgG as a blocking reagent for immune assays targeting bovine leukocytes in order to enhance the accuracy of the results.


Assuntos
Anticorpos Monoclonais/imunologia , Imunofenotipagem/métodos , Leucócitos Mononucleares/imunologia , Receptores Fc/imunologia , Erro Experimental , Animais , Bovinos , Epitopos/imunologia , Citometria de Fluxo , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunofenotipagem/normas , Camundongos , Ligação Proteica
18.
Artigo em Inglês | MEDLINE | ID: mdl-31255699

RESUMO

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.


Assuntos
Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Ligação Proteica , Transporte Proteico , Proteínas de Peixe-Zebra/antagonistas & inibidores
19.
Eur Biophys J ; 48(6): 559-568, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273390

RESUMO

According to the generalized conformational selection model, ligand binding involves the co-existence of at least two conformers with different ligand-affinities in a dynamical equilibrium. Conformational transitions between them should be guaranteed by intramolecular vibrational dynamics associated to each conformation. These motions are, therefore, related to the biological function of a protein. Positions whose mutations are found to alter these vibrations the most can be defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. In a previous study, we have shown that these positions are evolutionarily conserved. They correspond to buried aliphatic residues mostly localized in regular structured regions of the protein like ß-sheets and α-helices. In the present paper, we perform a network analysis of these key positions for a large dataset of paired protein structures in the ligand-free and ligand-bound form. We observe that networks of interactions between these key positions present larger and more integrated networks with faster transmission of the information. Besides, networks of residues result that are robust to conformational changes. Our results reveal that the conformational diversity of proteins seems to be guaranteed by a network of strongly interconnected key positions rather than individual residues.


Assuntos
Proteínas/química , Proteínas/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Vibração
20.
Pharm Res ; 36(9): 130, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264003

RESUMO

PURPOSE: An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest. METHODS: To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency. RESULTS: The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged. CONCLUSION: This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida de Alta Pressão , Imagem por Ressonância Magnética , Ligação Proteica , Conformação Proteica , Espectrometria de Massas em Tandem
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