Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32.452
Filtrar
1.
Front Immunol ; 11: 565278, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013929

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. For better understanding the viral spike (S) protein variations and its potential effects on the interaction with the host immune system and also in vaccine development, the cell epitopes, glycosylation profile and their changes during the global transmission course were characterized and compared with SARS-CoV for their glycosylation profile. We analyzed totally 7,813 sequences screened from 8,897 whole genome sequences on GISAID database up to April 26, and 18 S protein amino acid variations with relatively high frequency (≥10-3) were identified. A total of 228 sequences of variants had multiple variations, of note, most of them harboring the D614G mutation. Among the predicted 69 linear B cell epitopes, 175 discontinuous B cell epitopes and 41 cytotoxic T lymphocyte epitopes in the viral S protein, we found that the protein structure and its potential function of some sites changed, such as the linear epitope length shortened and discontinuous epitope disappeared of G476S. In addition, we detected 9 predicted N-glycosylation sites and 3 O-glycosylation sites unique to SARS-CoV-2, but no evidently observed variation of the glycan sites so far. Our findings provided an important snapshot of temporal and geographical distributions on SARS-CoV-2 S protein cell epitopes and glycosylation sites, which would be an essential basis for the selection of vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Sequenciamento Completo do Genoma
2.
Tumour Biol ; 42(9): 1010428320957506, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32914709

RESUMO

The development of the multidrug resistance phenotype is one of the major challenges faced in the treatment of cancer. The multidrug resistance phenotype is characterized by cross-resistance to drugs with different chemical structures and mechanisms of action. In this work, we hypothesized that the acquisition of resistance in cancer is accompanied by activation of the epithelial-to-mesenchymal transition process, where the tumor cell acquires a more mobile and invasive phenotype; a fundamental step in tumor progression and in promoting the invasion of other organs and tissues. In addition, it is known that atypical glycosylations are characteristic of tumor cells, being used as biomarkers. We believe that the acquisition of the multidrug resistance phenotype and the activation of epithelial-to-mesenchymal transition provoke alterations in the cell glycophenotype, which can be used as glycomarkers for chemoresistance and epithelial-to-mesenchymal transition processes. Herein, we induced the multidrug resistance phenotype in the PC-3 human prostate adenocarcinoma line through the continuous treatment with the drug paclitaxel. Our results showed that the induced cell multidrug resistance phenotype (1) acquired a mixed profile between epithelial and mesenchymal phenotypes and (2) modified the glycophenotype, showing an increase in the level of sialylation and in the number of branched glycans. Both mechanisms are described as indicators of poor prognosis.


Assuntos
Adenocarcinoma/patologia , Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Paclitaxel/farmacologia , Adenocarcinoma/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Glicosilação , Humanos , Células PC-3 , Fenótipo
3.
Nat Commun ; 11(1): 4646, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938911

RESUMO

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Hemaglutininas Virais/química , Proteínas Virais de Fusão/química , Betacoronavirus/classificação , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Glicosilação , Células HEK293 , Hemaglutininas Virais/metabolismo , Hemaglutininas Virais/ultraestrutura , Humanos , Lectinas/química , Lectinas/metabolismo , Espectrometria de Massas , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Domínios Proteicos , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/ultraestrutura
4.
Sci Rep ; 10(1): 14991, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929138

RESUMO

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the innate and adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the notable exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight of the S trimer (17% for the HEK293 glycoform) they shield approximately 40% of the protein surface.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Polissacarídeos/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Imunidade Adaptativa , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Glicosilação , Células HEK293 , Antígenos HLA/metabolismo , Humanos , Imunidade Inata , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
5.
Nat Commun ; 11(1): 4864, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978392

RESUMO

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal ß-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.


Assuntos
Tolerância a Medicamentos/fisiologia , Fungos/enzimologia , Fungos/metabolismo , Xilosidases/química , Xilosidases/metabolismo , Biocatálise , Domínio Catalítico , Fungos/efeitos dos fármacos , Glicoconjugados/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese , Especificidade por Substrato , Talaromyces/enzimologia , Talaromyces/genética , Xilosidases/genética
6.
Nat Commun ; 11(1): 4874, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978395

RESUMO

Organic synthesis methodology enables the synthesis of complex molecules and materials used in all fields of science and technology and represents a vast body of accumulated knowledge optimally suited for deep learning. While most organic reactions involve distinct functional groups and can readily be learned by deep learning models and chemists alike, regio- and stereoselective transformations are more challenging because their outcome also depends on functional group surroundings. Here, we challenge the Molecular Transformer model to predict reactions on carbohydrates where regio- and stereoselectivity are notoriously difficult to predict. We show that transfer learning of the general patent reaction model with a small set of carbohydrate reactions produces a specialized model returning predictions for carbohydrate reactions with remarkable accuracy. We validate these predictions experimentally with the synthesis of a lipid-linked oligosaccharide involving regioselective protections and stereoselective glycosylations. The transfer learning approach should be applicable to any reaction class of interest.


Assuntos
Carboidratos/química , Aprendizado de Máquina , Oligossacarídeos/química , Metabolismo dos Carboidratos , Técnicas de Química Sintética , Glicosilação , Estrutura Molecular , Oligossacarídeos/metabolismo
7.
J Phys Chem Lett ; 11(19): 8084-8093, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32885971

RESUMO

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.


Assuntos
Epitopos/química , Simulação de Dinâmica Molecular , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/química , Sítios de Ligação de Anticorpos , Glicosilação , Humanos , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Polissacarídeos/química
8.
Environ Pollut ; 265(Pt A): 114285, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32806420

RESUMO

BACKGROUND: Perfluorooctanoic acid (PFOA) and perfluorooctanoic sulfonate (PFOS) have been shown to be associated with disease development. Immunoglobulin G (IgG) N-glycosylation plays a vital role in human immune system and inflammatory activities. Altered IgG glycosylation was one of the molecular markers of various disorders. However, whether the chemicals affect IgG glycosylation has not been investigated. METHODS: Serum samples of 190 individuals including 95 adults and 95 children were selected based on the sex, age and PFOA/PFOS concentration. IgG N-glycome profile was obtained from glycan release, derivatization, and MALDI-MS analysis. One-factor ANOVA test was performed to analyze the association between different levels of PFOS/PFOA and IgG glycosylation changes. Evaluation of the diagnostic performance of significantly changed IgG glycosylation was performed by receiver operating characteristic curve. PFOS/PFOA concentrations were studied in relation to IgG glycosylation by 3D-nonlinear regression analysis. RESULTS: 10 of the 28 individual IgG glycans were significantly altered between different levels of PFOS/PFOA in adult serum. Among children with high serum levels of PFOS or PFOA, a total of 12 IgG N-glycans were markedly different from those with lower serum PFOS/PFOA. The glycan derived traits for adults with higher serum PFOS or PFOA were marked by significant alterations in IgG digalactosylation, agalactosylation, fucosylation, fucosylated sialylation, and disialylation. Similarly, pronounced changes in agalactosylation, digalactosylation, mono-sialylation and total sialylation, as well as neutral and sialo bisection, were associated with elevated serum PFOS or PFOA in children. Several glycans gained moderately accurate scores of area under the curve for diagnosis of PFOS or PFOA pollution. Nonlinear surface fitting showed the independent or coordinate effect of PFOS or PFOA on the expression of IgG glycosylation. CONCLUSIONS: High levels of PFOS or PFOA in human serum were strongly associated with altered IgG glycosylation and therefore are a potential risk factor for the development of diseases.


Assuntos
Ácidos Alcanossulfônicos , Adulto , Caprilatos , Criança , Exposição Ambiental , Fluorcarbonetos , Glicosilação , Humanos , Imunoglobulina G
9.
PLoS One ; 15(8): e0237295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756606

RESUMO

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.


Assuntos
Betacoronavirus/metabolismo , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glicosilação , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Nat Commun ; 11(1): 3990, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778659

RESUMO

The molecular mechanisms regulating lymphocyte homing into lymph nodes are only partly understood. Here, we report that B cell-specific deletion of the X-linked gene, Cosmc, and the consequent decrease of protein O-glycosylation, induces developmental blocks of mouse B cells. After transfer into wild-type recipient, Cosmc-null B cells fail to home to lymph nodes as well as non-lymphoid organs. Enzymatic desialylation of wild-type B cells blocks their migration into lymph nodes, indicating a requirement of sialylated O-glycans for proper trafficking. Mechanistically, Cosmc-deficient B cells have normal rolling and firm arrest on high endothelium venules (HEV), thereby attributing their inefficient trafficking to alterations in the subsequent transendothelial migration step. Finally, Cosmc-null B cells have defective chemokine signaling responses. Our results thus demonstrate that Cosmc and its effects on O-glycosylation are important for controlling B cell homing.


Assuntos
Linfócitos B/metabolismo , Linfonodos/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Movimento Celular , Feminino , Glicosilação , Humanos , Imunidade Humoral/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Polissacarídeos/metabolismo , Transcriptoma , Vênulas
11.
Nat Commun ; 11(1): 4033, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820167

RESUMO

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.


Assuntos
Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Idoso , Animais , Linhagem Celular , Desenho de Fármacos , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Ratos , Suínos
12.
Med Sci (Paris) ; 36(8-9): 735-746, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32821050

RESUMO

Glycosylation is an essential and complex cellular process where monosaccharides are added one by one onto an acceptor molecule, most of the time a protein or a lipid, so called glycoprotein or glycolipid. This cellular process is found in every living organism and is tightly conserved during evolution. In human, if one of the glycosylation reactions is genetically impaired, Congenital Disorders of Glycosylation (CDG) appear. CDG are a growing family of more than a hundred genetic diseases. This review offers a panorama of CDGs from 1980 to the present, their discoveries, diagnoses and treatments.


Assuntos
Defeitos Congênitos da Glicosilação , Animais , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/epidemiologia , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/terapia , Estudos de Associação Genética/história , Estudos de Associação Genética/tendências , Testes Genéticos/história , Testes Genéticos/métodos , Testes Genéticos/tendências , Glicosilação , História do Século XX , História do Século XXI , Humanos
13.
J Chromatogr A ; 1627: 461422, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823117

RESUMO

Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 µL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.


Assuntos
Glicopeptídeos/análise , Histidina/química , Ácido N-Acetilneuramínico/química , Sequência de Aminoácidos , Avidina/química , Fetuínas/química , Glicopeptídeos/sangue , Glicosilação , Humanos , Microesferas , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
J Transl Med ; 18(1): 281, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650788

RESUMO

BACKGROUND: The recent outbreak by SARS-CoV-2 has generated a chaos in global health and economy and claimed/infected a large number of lives. Closely resembling with SARS CoV, the present strain has manifested exceptionally higher degree of spreadability, virulence and stability possibly due to some unidentified mutations. The viral spike glycoprotein is very likely to interact with host Angiotensin-Converting Enzyme 2 (ACE2) and transmits its genetic materials and hijacks host machinery with extreme fidelity for self propagation. Few attempts have been made to develop a suitable vaccine or ACE2 blocker or virus-receptor inhibitor within this short period of time. METHODS: Here, attempt was taken to develop some therapeutic and vaccination strategies with a comparison of spike glycoproteins among SARS-CoV, MERS-CoV and the SARS-CoV-2. We verified their structure quality (SWISS-MODEL, Phyre2, and Pymol) topology (ProFunc), motifs (MEME Suite, GLAM2Scan), gene ontology based conserved domain (InterPro database) and screened several epitopes (SVMTrip) of SARS CoV-2 based on their energetics, IC50 and antigenicity with regard to their possible glycosylation and MHC/paratope binding (Vaxigen v2.0, HawkDock, ZDOCK Server) effects. RESULTS: We screened here few pairs of spike protein epitopic regions and selected their energetic, Inhibitory Concentration50 (IC50), MHC II reactivity and found some of those to be very good target for vaccination. A possible role of glycosylation on epitopic region showed profound effects on epitopic recognition. CONCLUSION: The present work might be helpful for the urgent development of a suitable vaccination regimen against SARS CoV-2.


Assuntos
Betacoronavirus/imunologia , Biologia Computacional/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Infecções por Coronavirus/prevenção & controle , Glicosilação , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Concentração Inibidora 50 , Anotação de Sequência Molecular , Pandemias , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/química
15.
Proc Natl Acad Sci U S A ; 117(32): 19399-19407, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719124

RESUMO

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética
16.
Nat Commun ; 11(1): 3424, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647180

RESUMO

We have determined the cryo-electron microscopic (cryo-EM) structures of two archaeal type IV pili (T4P), from Pyrobaculum arsenaticum and Saccharolobus solfataricus, at 3.8 Å and 3.4 Å resolution, respectively. This triples the number of high resolution archaeal T4P structures, and allows us to pinpoint the evolutionary divergence of bacterial T4P, archaeal T4P and archaeal flagellar filaments. We suggest that extensive glycosylation previously observed in T4P of Sulfolobus islandicus is a response to an acidic environment, as at even higher temperatures in a neutral environment much less glycosylation is present for Pyrobaculum than for Sulfolobus and Saccharolobus pili. Consequently, the Pyrobaculum filaments do not display the remarkable stability of the Sulfolobus filaments in vitro. We identify the Saccharolobus and Pyrobaculum T4P as host receptors recognized by rudivirus SSRV1 and tristromavirus PFV2, respectively. Our results illuminate the evolutionary relationships among bacterial and archaeal T4P filaments and provide insights into archaeal virus-host interactions.


Assuntos
Archaea/metabolismo , Proteínas Arqueais/química , Evolução Biológica , Sequência de Aminoácidos , Archaea/virologia , Proteínas Arqueais/ultraestrutura , Sequência Conservada , Glicosilação , Domínios Proteicos , Estrutura Secundária de Proteína
17.
PLoS One ; 15(7): e0235687, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678825

RESUMO

Lactobacillus amylolyticus L6, a gram-positive amylolytic bacterium isolated from naturally fermented tofu whey (NFTW), was able to hydrolyze raffinose and stachyose for the production of α-galactosidase. The cell-free extract of L. amylolyticus L6 was found to exhibit glycosyltransferase activity to synthesize α-galacto-oligosaccharides (GOS) with melibiose as substrate. The coding genes of α-galactosidase were identified in the genome of L. amylolyticus L6. The α-galactosidase (AglB) was placed into GH36 family by amino acid sequence alignments with other α-galactosidases from lactobacilli. The optimal reaction conditions of pH and temperature for AglB were pH 6.0 and 37°C, respectively. Besides, potassium ion was found to improve the activity of AglB while divalent mercury ion, copper ion and zinc ion displayed different degrees of inhibition effect. Under the optimum reaction condition, AglB could catalyze the synthesis of GOS with degree of polymerization (DP) ≥5 by using 300 mM melibiose concentration as substrate. The maximum yield of GOS with (DP) ≥3 could reach 31.56% (w/w). Transgalactosyl properties made AglB a potential candidate for application in the production of GOS.


Assuntos
Proteínas de Bactérias/metabolismo , Clonagem Molecular , Lactobacillus/enzimologia , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura , alfa-Galactosidase/química , alfa-Galactosidase/genética
18.
Chem Biol Interact ; 328: 109201, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32717190

RESUMO

The caseinate and glycated caseinate generated from the transglutaminase-catalyzed reaction of caseinate and oligochitosan were digested using pepsin and trypsin, and the activity of the resultant digests was measured in rat intestinal epithelial cell line (IEC-6) using several biological responses as indicators. Compared with the caseinate digest, the glycated caseinate digest had similar contents in 17 amino acids but less reactable -NH2 contents, and 6.57 g glucosamine per kg protein; moreover, it showed higher activity in the cells (P < 0.05) to promote cell growth, accumulate the cell-cycle progression at the S-phase, and prevent the camptothecin-induced cell apoptosis. The glycated caseinate digest also showed higher differentiation activity in the cells than the caseinate digest, resulting in enhanced activities of the three brush-border membrane enzymes (P < 0.05) and increased microvilli on the cell surfaces. The real-time reverse transcription-polymerase chain reaction, Western-blot assay, and Dickkopf-1 (a receptor inhibitor of the Wnt/ß-catenin signaling pathway) were used to determine both gene and protein expression changes in the cells. A Wnt/ß-catenin signaling pathway responsible for these enhanced effects was proposed because the five genes (glycogen synthase kinase 3ß, Wnt3a, ß-catenin, c-Myc, and cyclin D1) and three proteins (nuclear and cytosolic ß-catenin, cyclin D1, and c-Myc) as part of this signaling pathway were regulated in the treated cells. The oligochitosan glycation of caseinate induced by transglutaminase is thus suggested endowing the peptic-tryptic caseinate digest with higher activity in the cells through its effects on the Wnt/ß-catenin signaling pathway.


Assuntos
Caseínas/metabolismo , Quitina/análogos & derivados , Enterócitos/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Bovinos , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular , Quitina/metabolismo , Enterócitos/citologia , Enterócitos/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
19.
Science ; 369(6506): 1005-1010, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616672

RESUMO

Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo-electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease.


Assuntos
Infecções Urinárias/metabolismo , Uromodulina/química , Uromodulina/fisiologia , Adesinas Bacterianas/química , Microscopia Crioeletrônica , Glicosilação , Humanos , Ligantes
20.
Food Chem ; 332: 127302, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615389

RESUMO

The study aimed to evaluate the inhibitory effects of Centella asiatica phenolics (CAP) on bovine serum albumin glycoxidation in a BSA-glucose model in vitro. The impact of the phenolic extract on the formation of total fluorescent advanced glycation end products (AGEs) and Amadori adducts were determined. Dityrosine, N-formylkynurenine, kynurenine and protein-carbonyls were quantified as markers of protein oxidation. Protein structural perturbations were determined by Congo red binding and FTIR analysis. Chemical characterization and CAP phytoconstituent profile was obtained by colorimetric and UHPLC-ESI-qTOF-MS analysis, respectively. Our data show that CAP attenuated the formation of fluorescent AGEs (38.5%), Dityrosine (44.6%), N-formylkynurenine (42.9%), Amadori products, and resisted structural alterations of BSA subjected to glycation. These effects could be due to the antioxidant and radical scavenging activities of CAP mediated by the presence of phenolics and triterpenoids. The results collectively suggest that CAP possesses antiglycative properties with potentials for nutraceutical applications.


Assuntos
Antioxidantes/farmacologia , Centella/química , Glucose/metabolismo , Fenóis/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Glicosilação/efeitos dos fármacos , Oxirredução
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA