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1.
Food Chem ; 334: 127554, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32711267

RESUMO

In this study, the targeted regulatory mechanism of pulsed electric field (PEF) was explored for antioxidant activity improvement in four peptides, RGAVIH, RGAVLH, VNAVIH, and VNAVLH, of the pine nut (Pinus koraiensis Sieb. et Zucc). The VNAVLH peptide exhibited the best antioxidant activity and the ß-sheet content decreased to a minimum value at 40 kV/cm. Moreover, the chemical shifts of hydrogen atoms of 2-H Asn and 6-H His shifted to a higher magnetic field. The connectivity between NαH (3.62 ppm) and CαH (8.10 ppm) of 6-His residue disappeared in PEF-treated peptide. Molecule dynamics (MD) simulation verified that the distances of Nα(H78)-Cα(H80) and H82-O94 increased, whereas -OH and -Cß(H83) got closer in histidine residue after applying the electric field force. Therefore, the antioxidant activity enhancement of VNAVLH might due to the targeted regulation of PEF treatment on NαH-CαH and imidazole group in histidine.


Assuntos
Histidina/química , Nozes/metabolismo , Peptídeos/química , Pinus/metabolismo , Sequência de Aminoácidos , Antioxidantes/química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta
2.
BMC Bioinformatics ; 21(1): 503, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33153432

RESUMO

BACKGROUND: The formation of contacts among protein secondary structure elements (SSEs) is an important step in protein folding as it determines topology of protein tertiary structure; hence, inferring inter-SSE contacts is crucial to protein structure prediction. One of the existing strategies infers inter-SSE contacts directly from the predicted possibilities of inter-residue contacts without any preprocessing, and thus suffers from the excessive noises existing in the predicted inter-residue contacts. Another strategy defines SSEs based on protein secondary structure prediction first, and then judges whether each candidate SSE pair could form contact or not. However, it is difficult to accurately determine boundary of SSEs due to the errors in secondary structure prediction. The incorrectly-deduced SSEs definitely hinder subsequent prediction of the contacts among them. RESULTS: We here report an accurate approach to infer the inter-SSE contacts (thus called as ISSEC) using the deep object detection technique. The design of ISSEC is based on the observation that, in the inter-residue contact map, the contacting SSEs usually form rectangle regions with characteristic patterns. Therefore, ISSEC infers inter-SSE contacts through detecting such rectangle regions. Unlike the existing approach directly using the predicted probabilities of inter-residue contact, ISSEC applies the deep convolution technique to extract high-level features from the inter-residue contacts. More importantly, ISSEC does not rely on the pre-defined SSEs. Instead, ISSEC enumerates multiple candidate rectangle regions in the predicted inter-residue contact map, and for each region, ISSEC calculates a confidence score to measure whether it has characteristic patterns or not. ISSEC employs greedy strategy to select non-overlapping regions with high confidence score, and finally infers inter-SSE contacts according to these regions. CONCLUSIONS: Comprehensive experimental results suggested that ISSEC outperformed the state-of-the-art approaches in predicting inter-SSE contacts. We further demonstrated the successful applications of ISSEC to improve prediction of both inter-residue contacts and tertiary structure as well.


Assuntos
Algoritmos , Proteínas/química , Bases de Dados de Proteínas , Proteínas de Membrana/química , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína
3.
Molecules ; 25(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036293

RESUMO

A novel series of some hydrazones bearing thiazole moiety were generated via solvent-drop grinding of thiazole carbohydrazide 2 with various carbonyl compounds. Also, dehydrative-cyclocondensation of 2 with active methylene compounds or anhydrides gave the respective pyarzole or pyrazine derivatives. The structures of the newly synthesized compounds were established based on spectroscopic evidences and their alternative syntheses. Additionally, the anti-viral activity of all the products was tested against SARS-CoV-2 main protease (Mpro) using molecular docking combined with molecular dynamics simulation (MDS). The average binding affinities of the compounds 3a, 3b, and 3c (-8.1 ± 0.33 kcal/mol, -8.0 ± 0.35 kcal/mol, and -8.2 ± 0.21 kcal/mol, respectively) are better than that of the positive control Nelfinavir (-6.9 ± 0.51 kcal/mol). This shows the possibility of these three compounds to effectively bind to SARS-CoV-2 Mpro and hence, contradict the virus lifecycle.


Assuntos
Antivirais/síntese química , Betacoronavirus/enzimologia , Hidrazonas/síntese química , Inibidores de Proteases/síntese química , Pirazinas/síntese química , Pirazóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Humanos , Hidrazonas/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirazinas/farmacologia , Pirazóis/farmacologia , Termodinâmica , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
4.
PLoS One ; 15(9): e0232311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915778

RESUMO

Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Cadeias kappa de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica em Folha beta , Coelhos
5.
PLoS One ; 15(9): e0239836, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986761

RESUMO

Plants are constantly under the threat of various biotic and abiotic stress conditions and to overcome these stresses, they have evolved multiple mechanisms including systematic accumulation of different phytohormones, phytoalexins and pathogenesis related (PR) proteins. PR proteins are cluster of proteins with low molecular weight which get incited in plants under different stresses. In this paper, in-silico approaches are used to compare the physico-chemical properties of 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) of Arabidopsis thaliana and Oryza sativa. Topological analysis revealed the presence of transmembrane localization of PR2 and absence of transmembrane domain in PR10 of both model plants studied. Amino acid composition shows the dominance of small aliphatic amino acids i.e. alanine, glycine and serine in both plants studied. These results highlights the similarities and differences between PRs of both model plants, which provides clue towards their diversified roles in plants.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Simulação por Computador , Oryza/química , Sequência de Aminoácidos , Aminoácidos , Ponto Isoelétrico , Proteínas de Membrana/química , Peso Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos
6.
Molecules ; 25(18)2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927621

RESUMO

Mass spectrometry and some other biophysical methods, have made substantial contributions to the studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins interactions. The most interesting feature of SARS-CoV-2 seems to be the structure of its spike (S) protein and its interaction with the human cell receptor. Mass spectrometry of spike S protein revealed how the glycoforms are distributed across the S protein surface. X-ray crystallography and cryo-electron microscopy made huge impact on the studies on the S protein and ACE2 receptor protein interaction, by elucidating the three-dimensional structures of these proteins and their conformational changes. The findings of the most recent studies in the scope of SARS-CoV-2-Human protein-protein interactions are described here.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/epidemiologia , Receptores Virais/química , Síndrome Respiratória Aguda Grave/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/patogenicidade , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
Molecules ; 25(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867349

RESUMO

Three types of new coronaviruses (CoVs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and corona-virus disease 2019 (COVID-19). Neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. Each of the 3C-like (3CL) proteases of the three CoVs is essential for the proliferation of the CoVs, and an inhibitor of the 3CL protease (3CLpro) is thought to be an ideal therapeutic agent against SARS, MERS, or COVID-19. Among these, SARS-CoV is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. This article briefly reviews a series of studies on SARS-CoV, focusing on the development of inhibitors for the SARS-CoV 3CLpro based on molecular interactions with the 3CL protease. Our recent approach, based on the structure-based rational design of a novel scaffold for SARS-CoV 3CLpro inhibitor, is also included. The achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including SARS-CoV-2.


Assuntos
Antivirais/química , Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Inibidores de Proteases/química , Vírus da SARS/patogenicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/genética , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
8.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
9.
Molecules ; 25(17)2020 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-32842509

RESUMO

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the Informational spectrum method applied for small molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Mezlocilina/química , Inibidores de Proteases/química , Raltegravir Potássico/química , Proteínas não Estruturais Virais/química , Sítio Alostérico , Antivirais/farmacologia , Betacoronavirus/enzimologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Mezlocilina/farmacologia , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Raltegravir Potássico/farmacologia , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
10.
Molecules ; 25(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842606

RESUMO

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Catepsina L/química , Compostos Fitoquímicos/química , Inibidores de Proteases/química , Receptores Virais/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Sítios de Ligação , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Catepsina L/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cumarínicos/química , Cumarínicos/isolamento & purificação , Cumarínicos/farmacologia , Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Índia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monossacarídeos/química , Monossacarídeos/isolamento & purificação , Monossacarídeos/farmacologia , Pandemias , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Plantas Medicinais/química , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Termodinâmica , Internalização do Vírus/efeitos dos fármacos
11.
Biochem Biophys Res Commun ; 532(1): 134-138, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32829876

RESUMO

SARS-CoV-2 is the etiologic agent of COVID-19. There is currently no effective means of preventing infections by SARS-CoV-2, except through restriction of population movement and contact. An understanding of the origin, evolution and biochemistry (molecular biology) of SARS-CoV-2 is a prerequisite to its control. Mutations in the phosphorylation sites of SARS-CoV-2 encoded nucleocapsid protein isolated from various populations and locations, are described. Mutations occurred in the phosphorylation sites, all located within a stretch which forms a phosphorylation dependent interaction site, including C-TAK1 phosphorylation sites for 14-3-3. The consequences of these mutations are discussed and a structure-based model for the role of protein 14-3-3 in the sequestration and inhibition of SARS-CoV-2 nucleocapsid protein's function is presented. It is proposed that the phosphorylation of SARS-CoV-2 nucleocapsid protein and its sequestration by Protein 14-3-3 is a cellular response mechanism for the control and inhibition of the replication, transcription and packaging of the SARS-CoV-2 genome.


Assuntos
Proteínas 14-3-3/química , Betacoronavirus/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Proteínas do Nucleocapsídeo/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosforilação , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
12.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
14.
Nat Commun ; 11(1): 4141, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811830

RESUMO

Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.


Assuntos
Anticorpos Neutralizantes/química , Herpesvirus Humano 3/química , Proteínas do Envelope Viral/química , Internalização do Vírus , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Microscopia Crioeletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/ultraestrutura
15.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641474

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.


Assuntos
Anticorpos Neutralizantes/farmacologia , Citomegalovirus/genética , Proteínas Mutantes Quiméricas/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Fusão Celular , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/ultraestrutura , Células Gigantes/virologia , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/virologia , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo
16.
Virus Res ; 288: 198082, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621841

RESUMO

The outbreak of the 2019 novel coronavirus (SARS-CoV-2) has infected millions of people with a large number of deaths across the globe. The existing therapies are limited in dealing with SARS-CoV-2 due to the sudden appearance of the virus. Therefore, vaccines and antiviral medicines are in desperate need. We took immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein (S), membrane glycoprotein (M) and nucleocapsid protein (N) of SARS-CoV-2, followed by estimating their antigenicity and interactions with the human leukocyte antigen (HLA) alleles. Allergenicity, toxicity, physiochemical properties analysis and stability were examined to confirm the specificity and selectivity of the epitope candidates. We identified a total of five B cell epitopes in RBD of S protein, seven MHC class-I, and 18 MHC class-II binding T-cell epitopes from S, M and N protein which showed non-allergenic, non-toxic and highly antigenic features and non-mutated in 55,179 SARS-CoV-2 virus strains until June 25, 2020. The epitopes identified here can be a potentially good candidate repertoire for vaccine development.


Assuntos
Betacoronavirus/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Proteínas do Nucleocapsídeo/química , Glicoproteína da Espícula de Coronavírus/química , Proteínas da Matriz Viral/química , Vacinas Virais/química , Sequência de Aminoácidos , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Imunogenicidade da Vacina , Modelos Moleculares , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/biossíntese
17.
Virus Res ; 288: 198102, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717346

RESUMO

Coronavirus disease 2019 (COVID-19) is an infectious disease, caused by a newly emerged highly pathogenic virus called novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Targeting the main protease (Mpro, 3CLpro) of SARS-CoV-2 is an appealing approach for drug development because this enzyme plays a significant role in the viral replication and transcription. The available crystal structures of SARS-CoV-2 Mpro determined in the presence of different ligands and inhibitor-like compounds provide a platform for the quick development of selective inhibitors of SARS-CoV-2 Mpro. In this study, we utilized the structural information of co-crystallized SARS-CoV-2 Mpro for the structure-guided drug discovery of high-affinity inhibitors from the PubChem database. The screened compounds were selected on the basis of their physicochemical properties, drug-likeliness, and strength of affinity to the SARS-CoV-2 Mpro. Finally, we have identified 6-Deaminosinefungin (PubChem ID: 10428963) and UNII-O9H5KY11SV (PubChem ID: 71481120) as potential inhibitors of SARS-CoV-2 Mpro which may be further exploited in drug development to address SARS-CoV-2 pathogenesis. Both compounds are structural analogs of known antivirals, having considerable protease inhibitory potential with improved pharmacological properties. All-atom molecular dynamics simulations suggested SARS-CoV-2 Mpro in complex with these compounds is stable during the simulation period with minimal structural changes. This work provides enough evidence for further implementation of the identified compounds in the development of effective therapeutics of COVID-19.


Assuntos
Aminoglicosídeos/química , Antivirais/química , Betacoronavirus/química , Inibidores de Proteases/química , Pirrolidinas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Aminoglicosídeos/metabolismo , Antivirais/metabolismo , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirrolidinas/metabolismo , Especificidade por Substrato , Termodinâmica , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
18.
Int J Nanomedicine ; 15: 4311-4324, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606679

RESUMO

Purpose: By providing a stem cell microenvironment with particular bioactive constituents in vivo, synthetic biomaterials have been progressively successful in stem cell-based tissue regeneration by enhancing the engraftment and survival of transplanted cells. Designs with bioactive motifs to influence cell behavior and with D-form amino acids to modulate scaffold stability may be critical for the development and optimization of self-assembling biomimetic hydrogel scaffolds for stem cell therapy. Materials and Methods: In this study, we linked naphthalene (Nap) covalently to a short D-form peptide (Nap-DFDFG) and the C domain of insulin-like growth factor-1 (IGF-1C) as a functional hydrogel-based scaffolds, and we hypothesized that this hydrogel could enhance the therapeutic efficiency of human placenta-derived mesenchymal stem cells (hP-MSCs) in a murine acute kidney injury (AKI) model. Results: The self-assembling peptide was constrained into a classical ß-sheet structure and showed hydrogel properties. Our results revealed that this hydrogel exhibited increased affinity for IGF-1 receptor. Furthermore, cotransplantation of the ß-IGF-1C hydrogel and hP-MSCs contributed to endogenous regeneration post-injury and boosted angiogenesis in a murine AKI model, leading to recovery of renal function. Conclusion: This hydrogel could provide a favorable niche for hP-MSCs and thereby rescue renal function in an AKI model by promoting cell survival and angiogenesis. In conclusion, by covalently linking the desired functional groups to D-form peptides to create functional hydrogels, self-assembling ß-sheet peptide hydrogels may serve as a promising platform for tissue-engineering and stem cell therapy.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Hidrogéis/química , Fator de Crescimento Insulin-Like I/química , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Peptídeos/química , Lesão Renal Aguda/fisiopatologia , Animais , Materiais Biocompatíveis/química , Sobrevivência Celular , Feminino , Fibrose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrogéis/síntese química , Rim/patologia , Rim/fisiopatologia , Camundongos Transgênicos , Neovascularização Fisiológica , Placenta/citologia , Gravidez , Conformação Proteica em Folha beta , Domínios Proteicos
19.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32647346

RESUMO

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Peptidil Dipeptidase A/química , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Quirópteros/virologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Evolução Molecular , Furina/química , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
20.
Mol Cell ; 79(3): 406-415.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692975

RESUMO

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.


Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
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