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1.
Nat Med ; 25(9): 1402-1407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501610

RESUMO

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Epitopos de Linfócito T/imunologia , Esclerose Múltipla/tratamento farmacológico , Natalizumab/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Neutralizantes/química , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Integrina alfa4/antagonistas & inibidores , Integrina alfa4/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Conformação Proteica/efeitos dos fármacos , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502568

RESUMO

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.


Assuntos
Estabilidade Enzimática/efeitos dos fármacos , Metais/farmacologia , Fosfopiruvato Hidratase/genética , Conformação Proteica/efeitos dos fármacos , Catálise/efeitos dos fármacos , Dicroísmo Circular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Íons/química , Íons/farmacologia , Metais/química , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/isolamento & purificação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade
3.
J Chem Theory Comput ; 15(10): 5169-5174, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31476124

RESUMO

Aggregation of amyloid-ß (Aß) peptides is a crucial step in the progression of Alzheimer's disease (AD). Identifying aggregation inhibitors against AD has been a great challenge. We report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and scyllo-inositol, which are AD drug candidates currently under investigation. We show that both small molecules promote a conformational change of the Aß42 monomer toward a more collapsed phase through a nonspecific binding mechanism. This finding provides atomistic-level insights into designing potential drug candidates for future AD treatments.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Peptídeos beta-Amiloides/química , Sítios de Ligação/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química
4.
Chem Commun (Camb) ; 55(74): 11052-11055, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31453599

RESUMO

Parkinson's disease (PD), closely associated with the misfolding and aggregation of the neuronal protein α-synuclein (A-Syn), is a neurodegenerative disorder with no cure to date. Here, we show that the commercially available, inexpensive, aminoglycoside antibiotic kanamycin effectively inhibits both lipid-induced and solution-phase aggregation of A-Syn in vitro, pointing towards the prospective repurposing of kanamycin as a potential anti-PD drug.


Assuntos
Antibacterianos/farmacologia , Canamicina/farmacologia , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Linhagem Celular Tumoral , Humanos , Canamicina/química , Canamicina/metabolismo , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
5.
J Recept Signal Transduct Res ; 39(2): 99-105, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31282287

RESUMO

Alzheimer's is a neural disorder causing gradual loss in structure and function of nerve cell. To treat such disorders, c-Jun N-terminal Kinase (JNK) Pathway inhibitors were developed by representing chemical compounds that were used to inhibit the JNK signaling pathways. DLK is the stress sensor and implicating as regulatory factor in JNK pathway. Therefore, in the present investigation, pharmacophore screening was tried to identify the chemical compounds that involving inhibition of DLK proteins. To explore the pharmacophore region and mode of binding with DLK protein, N- (I H-pyrazol-3-y l) pyridin-2-aminer inhibitors were docked with DLK. Results reveal the information on the interaction mechanism of protein and ligand with chemical characteristics required to inhibit DLK protein. Such predicted information (AAAARH) was used as query to find out potential novel lead compounds sourced from public database. As an outcome of 65 compounds were listed based on the fitness score (2≥), and were subjected to glide HTVS.SP and XP. Best performing 5 lead compounds were shortlisted for dynamic simulations. This exhibited a constant RMSD over 20 ns of timescale.


Assuntos
Doença de Alzheimer/economia , Proteínas de Ligação ao Cálcio/química , Inibidores Enzimáticos/química , Proteínas de Membrana/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Piridinas/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Domínio Catalítico/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , Conformação Proteica/efeitos dos fármacos
6.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269656

RESUMO

Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide. The class A serine ß-lactamase BlaC confers Mycobacterium tuberculosis resistance to conventional ß-lactam antibiotics. As the primary mechanism of bacterial resistance to ß-lactam antibiotics, the expression of a ß-lactamase by Mycobacterium tuberculosis results in hydrolysis of the ß-lactam ring and deactivation of these antibiotics. In this study, we conducted protein X-ray crystallographic analysis of the inactivation of BlaC, upon exposure to the inhibitor bis(benzoyl) phosphate. Crystal structure data confirms that serine ß-lactamase is phosphorylated at the catalytic serine residue (Ser-70) by this phosphate-based inactivator. This new crystallographic evidence suggests a mechanism for phosphorylation of BlaC inhibition by bis(benzoyl) phosphate over acylation. Additionally, we confirmed that bis(benzoyl) phosphate inactivated BlaC in a time-dependent manner.


Assuntos
Mycobacterium tuberculosis/enzimologia , Organofosfatos/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Sequência de Aminoácidos , Benzoatos/química , Benzoatos/farmacologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Organofosfatos/química , Conformação Proteica/efeitos dos fármacos , Alinhamento de Sequência , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismo
7.
Nat Struct Mol Biol ; 26(7): 545-556, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270469

RESUMO

Monoamine transporters (MATs) regulate neurotransmission via the reuptake of dopamine, serotonin and norepinephrine from extra-neuronal regions and thus maintain neurotransmitter homeostasis. As targets of a wide range of compounds, including antidepressants, substances of abuse and drugs for neuropsychiatric and neurodegenerative disorders, their mechanism of action and their modulation by small molecules have long been of broad interest. Recent advances in the structural characterization of dopamine and serotonin transporters have opened the way for structure-based modeling and simulations, which, together with experimental data, now provide mechanistic understanding of their transport function and interactions. Here we review recent progress in the elucidation of the structural dynamics of MATs and their conformational landscape and transitions, as well as allosteric regulation mechanisms.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Descoberta de Drogas , Humanos , Modelos Moleculares , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/química , Conformação Proteica/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/química
8.
Biosci Biotechnol Biochem ; 83(11): 2027-2033, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31322484

RESUMO

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression. Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp.


Assuntos
Colágeno/química , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos
9.
J Recept Signal Transduct Res ; 39(2): 154-166, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31355691

RESUMO

Asbtract Filamentous temperature-sensitive protein Z (FtsZ), playing a key role in bacterial cell division, is regarded as a promising target for the design of antimicrobial agent. This study is looking for potential high-efficiency FtsZ inhibitors. Ligand-based pharmacophore and E-pharmacophore, virtual screening and molecular docking were used to detect promising FtsZ inhibitors, and molecular dynamics simulation was used to study the stability of protein-ligand complexes in this paper. Sixty-three inhibitors from published literatures with pIC50 ranging from 2.483 to 5.678 were collected to develop ligand-based pharmacophore model. 4DXD bound with 9PC was selected to develop the E-pharmacophore model. The pharmacophore models validated by test set method and decoy set were employed for virtual screening to exclude inactive compounds against ZINC database. After molecular docking, ADME analysis, IFD docking and MM-GBSA, 8 hits were identified as potent FtsZ inhibitors. A 50 ns molecular dynamics simulation was implemented on the compounds to assess the stability between potent inhibitors and FtsZ. The results indicated that the candidate compounds had a high docking score and were strongly combined with FtsZ by forming hydrogen bonding interactions with key amino acid residues, and van der Waals forces and hydrophobic interactions had significant contribution to the stability of the binding. Molecular dynamics simulation results showed that the protein-ligand compounds performed well in both the stability and flexibility of the simulation process.


Assuntos
Anti-Infecciosos/química , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Conformação Proteica/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Cristalografia por Raios X , Proteínas do Citoesqueleto/antagonistas & inibidores , Humanos , Ligações de Hidrogênio/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Temperatura Ambiente
11.
Int J Biol Macromol ; 138: 800-809, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351960

RESUMO

Herein, we explored the interaction of Al2O3 NPs with RBCs and Hb to determine the effect of Al2O3 NPs on hemolytic activity and Hb denaturation. The percentage of hemolysis of extracts and direct contact assays triggered by Al2O3 NPs was calculated by determining supernatant Hb concentration at 540 nm. Far-UV CD and Trp/ANS/acrylamide fluorescence spectroscopic methods were used to determine the structural changes of Hb upon interaction with Al2O3 NPs. Theoretical studies were carried out to display the residues involved in the binding site of Hb with Al2O3 nanocluster as well as the structural changes of Hb after interaction. The results showed that the percentage of hemolysis of extract and direct contact assays induced by Al2O3 NPs were 1.16 and 0.46, respectively. Fluorescence spectroscopy revealed that Al2O3 NPs alter the quaternary structure of the protein; however, CD spectroscopy indicated that the secondary structure of Hb remains almost unchanged. Theoretical study displayed that Al2O3 nanocluster interacts with different residues of protein, and Hb tends to be destabilized at the binding site with nanocluster. This study may be significant in exploring the toxicity profile of Al2O3 NPs for their in vivo implementations.


Assuntos
Óxido de Alumínio/química , Óxido de Alumínio/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemoglobinas/química , Conformação Proteica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Análise Espectral
12.
Int J Biol Macromol ; 138: 168-180, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31306707

RESUMO

Polyphenols have been suggested as potential therapeutic agents for the treatment of amyloidogenic diseases. In this work, we evaluate quercetin-rich onion extract for its ability to inhibit tau fibrillization. Considering the presence of polyphenols in multiple glycosidic and aglycosidic forms, a nanobiocatalyst-mediated approach has been used to extract quercetin from onion skins. The nanobiocatalysts facilitate greater release of quercetin compared to the use of free enzymes. Atomic force microscopy and fluorescence microscopy show that quercetin possesses a novel inhibitory character on tau-fibril aggregation. In contrast, quercetin-diglucoside does not have an inhibitory effect. Molecular Dynamics simulations reveal conformational changes in tau protein upon interaction with quercetin due to specific hydrogen bonding and hydrophobic interactions. The resulting conformational stability of tau monomer reduces propensity of the protein to aggregate. The ability of quercetin to inhibit tau fibrillization expands the paradigm for application of bioactive polyphenols.


Assuntos
Biocatálise , Enzimas Imobilizadas/metabolismo , Nanopartículas/química , Nanoestruturas/química , Agregados Proteicos/efeitos dos fármacos , Quercetina/farmacologia , Proteínas tau/química , Enzimas Imobilizadas/química , Compostos Férricos/química , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Quercetina/química
13.
Chemosphere ; 234: 513-519, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31229712

RESUMO

Given the ubiquity of iodinated disinfection by-products (I-DBPs) in drinking water and their prominent toxicity, it is of vital significance to evaluate I-DBPs toxicity and explore the underlying mechanism. The toxicity of iodoacetic acid (IAA), a typical type of I-DBPs, might be linked with oxidative stress. However, it remains unknown for the response of antioxidant enzyme superoxide dismutase (SOD) in the mouse primary hepatocytes when exposed to IAA and the underlying mechanism. This study explored SOD response to IAA and the underlying mechanisms at the molecular and cellular levels. Under IAA exposure, the observed increase of SOD activity in the hepatocytes was caused by the increase of SOD production via ROS stimulation and the increase of SOD molecular activity. Molecular experiments showed that IAA binds to SOD molecule mainly via electrostatic forces with one binding site around the active site and six binding sites in the surface of protein. The binding interaction leads to the conformational changes of SOD and the disruption of protein aggregates. This work could offer basic data for the comprehensive understanding of the adverse effects of IAA and references for assessing the harmful effects of DBPs.


Assuntos
Desinfecção/métodos , Ácido Iodoacético/química , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/farmacologia , Água Potável/química , Hepatócitos/enzimologia , Ácido Iodoacético/metabolismo , Ácido Iodoacético/toxicidade , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos
14.
Nat Struct Mol Biol ; 26(7): 599-606, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235912

RESUMO

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.


Assuntos
Adenosina/metabolismo , Dilazep/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/química , Tioinosina/análogos & derivados , Cristalografia por Raios X , Dilazep/química , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Tioinosina/química , Tioinosina/farmacologia
15.
Int J Food Microbiol ; 305: 108240, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31202151

RESUMO

The lantibiotic nisin is used as a food additive to effectively inactivate a broad spectrum of Gram-positive bacteria such as Listeria monocytogenes. In total, 282 L. monocytogenes field isolates from German ready-to-eat food products, food-processing environments and patient samples and 39 Listeria reference strains were evaluated for their susceptibility to nisin. The MIC90 value was <1500 IU ml-1. Whole genome sequences (WGS) of four nisin susceptible (NS; growth <200 IU ml-1) and two nisin resistant L. monocytogenes field isolates (NR; growth >1500 IU ml-1) of serotype IIa were analyzed for DNA sequence variants (DSVs) in genes putatively associated with NR and its regulation. WGS of NR differed from NS in the gadD2 gene encoding for the glutamate decarboxylase system (GAD). Moreover, homology modeling predicted a protein structure of GadD2 in NR that promoted a less pH dependent GAD activity and may therefore be beneficial for nisin resistance. Likewise NR had a significant faster growth rate compared to NS in presence of nisin at pH 7. In conclusion, results contributed to ongoing debate that a genetic shift in GAD supports NR state.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Glutamato Descarboxilase/química , Listeria monocytogenes/efeitos dos fármacos , Nisina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Fast Foods/microbiologia , Aditivos Alimentares/farmacologia , Manipulação de Alimentos/métodos , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/metabolismo , Conformação Proteica/efeitos dos fármacos , Sequenciamento Completo do Genoma
16.
Int J Biol Macromol ; 136: 512-520, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199971

RESUMO

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site.


Assuntos
Cardiotoxinas/farmacologia , Quimotripsina/metabolismo , Naja naja , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cardiotoxinas/química , Cardiotoxinas/metabolismo , Quimotripsina/química , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos
17.
Eur Biophys J ; 48(6): 503-511, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31222413

RESUMO

A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.


Assuntos
Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Proteínas Secretadas pela Vesícula Seminal/farmacologia , Animais , Bovinos , Cães , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Hemaglutininas Virais/química , Vírus da Influenza A Subtipo H3N2/fisiologia , Células Madin Darby de Rim Canino , Fosforilcolina/farmacologia , Conformação Proteica/efeitos dos fármacos , Proteínas Secretadas pela Vesícula Seminal/metabolismo , Internalização do Vírus/efeitos dos fármacos
18.
Eur J Pharmacol ; 855: 183-191, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31082368

RESUMO

Myocardial fibrosis is associated with cardiovascular remodeling, which is characterized by abnormal collagen architecture. However, there are not yet effective strategies targeting this abnormal pathological process. The purpose of our study is to investigate the effect of protocatechualdehyde (PCA) on myocardial fibrosis for exploring the underlying target protein and molecular mechanism. We found PCA significantly suppressed isoprenaline (ISO)-induced fibrosis and collagen deposition in myocardial tissue. Then, the direct pharmacological target of PCA was identified as collagen I using cellular thermal shift assay (CETSA) coupled with stable isotope labeling with amino acids in cell culture (SILAC) technology. Surface plasmon resonance (SPR) analysis further confirmed the specific binding of PCA with collagen I. Moreover, collagen self-assembly assay and atomic force microscope analysis confirmed that PCA directly modulated collagen conformational dynamics. LC-MS/MS analysis was applied to determine lysine residues as the binding sites of PCA on collagen I by covalently cross-linking reaction. Collectively, our study suggests that PCA controls cardiovascular remodeling by mediating diffuse interstitial myocardial fibrosis. Moreover, directly targeting collagen may be a promising strategy for the treatment of heart failure and resultant myocardial fibrosis.


Assuntos
Benzaldeídos/farmacologia , Catecóis/farmacologia , Colágeno/química , Miocárdio/patologia , Animais , Linhagem Celular , Fibrose , Insuficiência Cardíaca/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Conformação Proteica/efeitos dos fármacos
19.
Spectrochim Acta A Mol Biomol Spectrosc ; 219: 313-318, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31054495

RESUMO

The interactions between cetyltrimethylammonium bromide (CTAB) and hen egg white lysozymes (HEWL) was carried out to investigate protein-surfactant interaction mechanisms while both exist in the overall same charged state. The interactions between CTAB and the HEWL were examined with circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and computational docking at a pH9.0 at room temperature. The far-UV CD and fluorescence results revealed that CTAB at concentrations from 0.15 to 10.0mM influenced the secondary as well as the tertiary structure of HEWL. The secondary structure of the HEWL was retained, while the tertiary structure of the HEWL was disrupted in the CTAB-treated samples at pH9.0. The hydrodynamic radii of the HEWL were also expanded in the presence of CTAB. Molecular docking studies showed that CTAB formed one electrostatic and four hydrophobic interactions, as well as one carbon hydrogen bond with HEWL. The data obtained from spectroscopic and computational studies demonstrated that the positively charged head and 18­carbon alkyl chain of the CTAB interacted through weak electrostatic and strong hydrophobic interactions.


Assuntos
Cetrimônio/metabolismo , Muramidase/metabolismo , Tensoativos/metabolismo , Animais , Galinhas , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Simulação de Acoplamento Molecular , Muramidase/química , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Eletricidade Estática
20.
Int J Biol Macromol ; 134: 344-353, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31075333

RESUMO

High glucose can lead to toxicity on islet ß cells. The protective effects of a novel Lentinus edodes mycelia polysaccharide (LMP) on INS-1 cells damaged by glucose were investigated. Cell viability, lactate dehydrogenase (LDH) release, cell apoptosis, intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were detected. P38 MAPK, JNKand NF-κB pathways were analyzed to reveal the inhibitory mechanism of LMP on glucose-induced INS-1 cells toxicity. The results showed that LMP could decrease cellular oxidative stress, reduce intracellular ROS levels, decrease MDA content and increase SOD activity. Furthermore, the glucose-induced cell apoptosis in cells were inhibited by regulating the expression of Bax, Bcl-2, cleaved caspase­3 and cleaved caspase­1. Cell signaling pathway analysis revealed that LMP could inhibit the activation of p38 MAPK, JNK, NF-κB pathways and activate Nrf2 pathway. To further explore the possible transportation mechanism of LMP with human serum albumin (HSA), ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy were used to evaluate the interaction between LMP and HSA. The results showed that LMP-HSA complex was formed, which would be helpful for explaining the transportation mechanism in vivo. These results suggested that LMP might be a new therapeutic candidate to alleviate the high glucose toxicity.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Polissacarídeos/metabolismo , Polissacarídeos/farmacologia , Albumina Sérica Humana/metabolismo , Cogumelos Shiitake/química , Apoptose/efeitos dos fármacos , Transporte Biológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Malondialdeído/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Albumina Sérica Humana/química , Superóxido Dismutase/metabolismo
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