Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
Bioconjug Chem ; 29(6): 1834-1840, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29723473

RESUMO

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP-peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol-ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA ( Ki = 1.3 µM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Simulação de Acoplamento Molecular , N-Acetilglucosaminiltransferases/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia
3.
Nature ; 556(7701): 381-385, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29643511

RESUMO

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biocatálise , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Cisteína/metabolismo , Esterificação , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteômica , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
4.
ACS Chem Biol ; 13(5): 1353-1360, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29641181

RESUMO

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.


Assuntos
Polarização de Fluorescência/métodos , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismo , Cristalografia por Raios X , Humanos , N-Acetilglucosaminiltransferases/química , Estudo de Prova de Conceito , Conformação Proteica , Especificidade por Substrato
5.
Chemistry ; 24(28): 7264-7272, 2018 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-29513364

RESUMO

A series of glycomimetics of UDP-GlcNAc, in which the ß-phosphate has been replaced by either an alkyl chain or a triazolyl ring and the sugar moiety has been replaced by a pyrrolidine ring, has been synthesized by the application of different click-chemistry procedures. Their affinities for human O-GlcNAc transferase (hOGT) have been evaluated and studied both spectroscopically and computationally. The binding epitopes of the best ligands have been determined in solution by means of saturation transfer difference (STD) NMR spectroscopy. Experimental, spectroscopic, and computational results are in agreement, pointing out the essential role of the binding of ß-phosphate. We have found that the loss of interactions from the ß-phosphate can be counterbalanced by the presence of hydrophobic groups at a pyrroline ring acting as a surrogate of the carbohydrate unit. Two of the prepared glycomimetics show inhibition at a micromolar level.


Assuntos
N-Acetilglucosaminiltransferases/química , Evolução Biológica , Simulação por Computador , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , N-Acetilglucosaminiltransferases/metabolismo
6.
ACS Chem Biol ; 12(8): 2078-2084, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28609614

RESUMO

O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner.


Assuntos
Acetilglucosamina/química , Espectrometria de Massas , Proteínas/metabolismo , Acilação , Glicosilação , Cinética , Processamento de Proteína Pós-Traducional , Proteínas/química
7.
Open Biol ; 7(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28659383

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.


Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Repetições de Tetratricopeptídeos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Glicosilação , Humanos , N-Acetilglucosaminiltransferases/genética , Alinhamento de Sequência , Especificidade por Substrato , Repetições de Tetratricopeptídeos/genética
8.
Biochem Soc Trans ; 44(1): 88-93, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862193

RESUMO

The O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove sugar moieties respectively. OGT knockout is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc and the dissection of catalytic compared with non-catalytic roles of OGT. Therefore, selective and potent chemical tools are necessary to inhibit OGT activity in the context of biological systems. The present review focuses on the available OGT inhibitors and summarizes advantages, limitations and future challenges.


Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , N-Acetilglucosaminiltransferases/metabolismo , Especificidade por Substrato/efeitos dos fármacos
9.
Nat Struct Mol Biol ; 22(9): 744-750, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26237509

RESUMO

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.


Assuntos
Domínio Catalítico , Glicosilação , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Cristalografia por Raios X , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
10.
Biochem J ; 457(3): 497-502, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24256146

RESUMO

Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Oligopeptídeos/farmacologia , Difosfato de Uridina/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Interferometria , Cinética , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/química , Difosfato de Uridina/química , Difosfato de Uridina/metabolismo , Difosfato de Uridina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA