RESUMO
Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Epitopos Imunodominantes/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Coronavirus/classificação , Coronavirus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/química , Reações Cruzadas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígeno HLA-B7/química , Antígeno HLA-B7/genética , Antígeno HLA-B7/imunologia , Humanos , Epitopos Imunodominantes/química , Memória Imunológica , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.
Assuntos
Alelos , Infecções Assintomáticas , COVID-19 , Antígenos HLA-B , Humanos , COVID-19/genética , COVID-19/imunologia , COVID-19/fisiopatologia , COVID-19/virologia , Epitopos de Linfócito T/imunologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Antígenos HLA-B/imunologia , Estudos de Coortes , Linfócitos T/imunologia , Epitopos Imunodominantes/imunologia , Reações Cruzadas/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Understanding the basis of the immune determinants controlling disease outcome is critical to provide better care to patients and could be exploited for therapeutics and vaccine design. The discovery of the human immunodeficiency virus (HIV) virus as the causing agent of acquired immunodeficiency syndrome (AIDS) decades ago, led to a tremendous amount of research. Among the findings, it was discovered that some rare HIV+ individuals, called HIV controllers (HICs), had the ability to control the virus and keep a low viral load without the need of treatment. This ability allows HICs to delay or avoid progression to AIDS. HIV control is strongly associated with the expression of human leukocyte antigen (HLA) alleles in HICs. From the HIV protective HLAs described, HLA-B57 is the most frequent in HIC patients. HLA-B57 can present a large range of highly conserved Gag-derived HIV peptides to CD8+ T cells and natural killer (NK) cells, both the focus of this review. So far there are limited differences in the immune response strength, magnitude, or receptor repertoire towards HIV epitopes that could explain viral control in HICs. Interestingly, some studies revealed that during early infection the large breadth of the immune response towards HIV mutants in HLA-B57+ HIC patients, might in turn influence the disease outcome.
Assuntos
Síndrome da Imunodeficiência Adquirida , Infecções por HIV , HIV-1 , Humanos , Antígenos HLA-B/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Matadoras Naturais/metabolismoRESUMO
The poly(A) tail at the 3' end of mRNAs determines their stability, translational efficiency, and fate. The shortening of the poly(A) tail, and its efficient removal, triggers the degradation of mRNAs, thus, regulating gene expression. The process is catalyzed by a family of enzymes, known as deadenylases. As the dysregulation of gene expression is a hallmark of cancer, understanding the role of deadenylases has gained additional interest. Herein, the genetic association network shows that CNOT6 and CNOT7 are the most prevalent and most interconnected nodes in the equilibrated diagram. Subsequent silencing and transcriptomic analysis identifies transcripts possibly regulated by specific deadenylases. Furthermore, several gene ontologies are enriched by common deregulated genes. Given the potential concerted action and overlapping functions of deadenylases, we examined the effect of silencing a deadenylase on the remaining ones. Our results suggest that specific deadenylases target unique subsets of mRNAs, whilst at the same time, multiple deadenylases may affect the same mRNAs with overlapping functions.
Assuntos
Carcinoma , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
As a major arm of the cellular immune response, CD4+ T cells are important in the control and clearance of infections. Primarily described as helpers, CD4+ T cells play an integral role in the development and activation of B cells and CD8+ T cells. CD4+ T cells are incredibly heterogeneous, and can be divided into six main lineages based on distinct profiles, namely T helper 1, 2, 17 and 22 (Th1, Th2, Th17, Th22), regulatory T cells (Treg) and T follicular helper cells (Tfh). Recent advances in structural biology have allowed for a detailed characterisation of the molecular mechanisms that drive CD4+ T cell recognition. In this review, we discuss the defining features of the main human CD4+ T cell lineages and their role in immunity, as well as their structural characteristics underlying their detection of pathogens.
Assuntos
Receptores de Antígenos de Linfócitos T/química , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Reações Antígeno-Anticorpo , Humanos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
The interaction between T cell receptor (TCR) and peptide (p)-Human Leukocyte Antigen (HLA) complexes is the critical first step in determining T cell responses. X-ray crystallographic studies of pHLA in TCR-bound and free states provide a structural perspective that can help understand T cell activation. These structures represent a static "snapshot", yet the nature of pHLAs and their interactions with TCRs are highly dynamic. This has been demonstrated for HLA class I molecules with in silico techniques showing that some interactions, thought to stabilise pHLA-I, are only transient and prone to high flexibility. Here, we investigated the dynamics of HLA class II molecules by focusing on three allomorphs (HLA-DR1, -DR11 and -DR15) that are able to present the same epitope and activate CD4+ T cells. A single TCR (F24) has been shown to recognise all three HLA-DR molecules, albeit with different affinities. Using molecular dynamics and crystallographic ensemble refinement, we investigate the molecular basis of these different affinities and uncover hidden roles for HLA polymorphic residues. These polymorphisms were responsible for the widening of the antigen binding cleft and disruption of pHLA-TCR interactions, underpinning the hierarchy of F24 TCR binding affinity, and ultimately T cell activation. We expanded this approach to all available pHLA-DR structures and discovered that all HLA-DR molecules were inherently rigid. Together with in vitro protein stability and peptide affinity measurements, our results suggest that HLA-DR1 possesses inherently high protein stability, and low HLA-DM susceptibility.
Assuntos
Antígenos/química , Antígenos HLA-DR/química , Receptores de Antígenos de Linfócitos T/química , Antígenos/imunologia , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Cristalografia por Raios X , Células HEK293 , Antígenos HLA-DR/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Human liver glycogen phosphorylase (hlGP), a key enzyme in glycogen metabolism, is a valid pharmaceutical target for the development of new anti-hyperglycaemic agents for type 2 diabetes. Inhibitor discovery studies have focused on the active site and in particular on glucopyranose based compounds with a ß-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the ß-pocket. Recently, C-ß-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the ß-pocket by studying the inhibitory effect of six different groups at the para position of 3-(ß-d-glucopyranosyl phenyl)-5-phenyl-, 1, 2, 4-triazoles in hlGP by kinetics and X-ray crystallography. The most bioactive compound was the one with an amine substituent to show a Ki value of 0.43⯵M. Structural studies have revealed the physicochemical diversity of the ß-pocket providing information for future rational inhibitor design studies.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Fígado/enzimologia , Triazóis/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicogênio Fosforilase/isolamento & purificação , Glicogênio Fosforilase/metabolismo , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Coelhos , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Human Angiogenin (hAng) is a member of the ribonuclease A superfamily and a potent inducer of neovascularization. Protein interactions of hAng in the nucleus and cytoplasm of the human umbilical vein cell line EA.hy926 have been investigated by mass spectroscopy. Data are available via ProteomeXchange with identifiers PXD006583 and PXD006584. The first gel-free analysis of hAng immunoprecipitates revealed many statistically significant potential hAng-interacting proteins involved in crucial biological pathways. Surprisingly, proliferating cell nuclear antigen (PCNA), was found to be immunoprecipitated with hAng only in the cytoplasm. The hAng-PCNA interaction and colocalization in the specific cellular compartment was validated with immunoprecipitation, immunoblotting, and immunocytochemistry. The results revealed that PCNA is predominantly localized in the cytoplasm, while hAng is distributed both in the nucleus and in the cytoplasm. hAng and PCNA colocalize in the cytoplasm, suggesting that they may interact in this compartment.
Assuntos
Citoplasma/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Proteômica , Ribonuclease Pancreático/metabolismo , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Humanos , Imunoprecipitação , Ligação Proteica/genética , Ribonuclease Pancreático/genéticaRESUMO
3-(C-Glucopyranosyl)-5aryl-1,2,4-triazoles with an aryl moiety larger than phenyl have been shown to have strong inhibitory potency (Ki values in the range of upper nM) for human liver glycogen phosphorylase (hlGP), a pharmacologically relevant target for diabetes type 2. In this study we investigate in a comparative manner the inhibitory effect of the above triazoles and their respective imidazoles on hlGPa. Kinetic studies show that the imidazole derivatives are 6-8 times more potent than their corresponding triazoles. We also seek to answer how the type of the aryl moiety affects the potency in hlGPa, and by determination of the crystal structure of rmGPb in complex with the triazole derivatives the structural basis of their inhibitory efficacy is also elucidated. Our studies revealed that the van der Waals interactions between the aryl moiety and residues in a hydrophobic pocket within the active site are mainly responsible for the variations in the potency of these inhibitors.
Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Triazóis/farmacologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/farmacologia , Cinética , Fígado/enzimologiaRESUMO
Eosinophil derived neurotoxin (EDN) is an eosinophil secretion protein and a member of the Ribonuclease A (RNase A) superfamily involved in the immune response system and inflammatory disorders. The pathological actions of EDN are strongly dependent on the enzymatic activity and therefore, it is of significant interest to discover potent and specific inhibitors of EDN. In this framework we have assessed the inhibitory potency of triazole double-headed ribonucleosides. We present here an efficient method for the heterologous production and purification of EDN together with the synthesis of nucleosides and their biochemical evaluation in RNase A and EDN. Two groups of double-headed nucleosides were synthesized by the attachment of a purine or a pyrimidine base, through a triazole group at the 3'-C position of a pyrimidine or a purine ribonucleoside, respectively. Based on previous data with mononucleosides these compounds were expected to improve the inhibitory potency for RNase A and specificity for EDN. Kinetics data revealed that despite the rational, all but one, double-headed ribonucleosides were less potent than the respective mononucleosides while they were also more specific for ribonuclease A than for EDN. Compound 11c (9-[3'-[4-[(cytosine-1-yl)methyl]-1,2,3-triazol-1-yl]-ß-d-ribofuranosyl]adenine) displayed a stronger preference for EDN than for ribonuclease A and a Ki value of 58µM. This is the first time that an inhibitor is reported to have a better potency for EDN than for RNase A. The crystal structure of EDN-11c complex reveals the structural basis of its potency and selectivity providing important guidelines for future structure-based inhibitor design efforts.
Assuntos
Eosinófilos/química , Neurotoxinas/antagonistas & inibidores , Ribonuclease Pancreático/antagonistas & inibidores , Ribonucleosídeos/farmacologia , Triazóis/farmacologia , Animais , Bovinos , Relação Dose-Resposta a Droga , Cinética , Modelos Moleculares , Estrutura Molecular , Neurotoxinas/metabolismo , Ribonuclease Pancreático/metabolismo , Ribonucleosídeos/química , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 N-acyl-ß-d-glucopyranosylamines putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a 'consensus scoring' approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants' values, in vitro, ranged from 5 to 377µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Glicogênio Fosforilase Hepática/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
HIV controllers can control viral replication and remain healthy, but the mechanism behind this control is unknown. Despite human leukocyte antigen (HLA) diversity in the population, almost 50% of HIV controllers express the HLA-B∗57:01 molecule, which presents, among others, the Gag-derived epitope TW10. Given TW10's presentation in early infection, TW10-specific T cells could participate in the control of HIV. Here, we study the strength and functionality of TW10-specific T cells from HLA-B∗57:01+/HIV+ controller and non-controller individuals. We determine the TW10-specific T cell receptor (TCR) repertoire, revealing a bias in TCR gene usage with the presence of a public TCR. We determine that the T cell response is polyfunctional regardless of the viral load, despite the low affinity of TW10-specific TCRs. We solve the crystal structure of HLA-B∗57:01-TW10 in complex with a TCR, providing the basis of recognition that underpins the strong TRBV5 bias observed in TW10-specific clonotypes.
Assuntos
Infecções por HIV , Antígenos HLA-B , Carga Viral , Humanos , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , HIV-1/imunologia , Masculino , Células ClonaisRESUMO
CD8+ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S976-984. We identified a longer and overlapping immunogenic peptide (S975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8+ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.
RESUMO
Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low µM inhibition constant (K(i)) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(ß-D-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with K(i) = 1.6 µM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B(1) subsite while the triazole moiety binds at the main subsite P(1), where P-O5' bond cleavage occurs, and the ribose at the interface between subsites P(1) and P(0) exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A-ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.
Assuntos
Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/farmacologia , Ribonuclease Pancreático/antagonistas & inibidores , Triazóis/química , Triazóis/farmacologia , Animais , Bovinos , Cristalografia por Raios X , Desenho de Fármacos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Nucleosídeos de Pirimidina/síntese química , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Triazóis/síntese químicaRESUMO
Understanding T-cell responses requires identifying viral peptides presented by human leukocyte antigens (HLAs). X-ray crystallography can be used to visualize their presentation. This protocol describes the expression, purification, and crystallization of HLA-A∗02:01, one of the most frequent HLA in the global population in complex with peptides derived from the SARS-CoV-2 nucleocapsid protein. This protocol can be applied to different HLA class I molecules bound to other peptides. For complete details on the use and execution of this protocol, please refer to Szeto et al. (2021).
Assuntos
COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Antígeno HLA-A2/química , Fragmentos de Peptídeos/química , SARS-CoV-2/metabolismo , Linfócitos T/imunologia , COVID-19/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismoRESUMO
CD8+ T cells are crucial for anti-viral immunity; however, understanding T cell responses requires the identification of epitopes presented by human leukocyte antigens (HLA). To date, few SARS-CoV-2-specific CD8+ T cell epitopes have been described. Internal viral proteins are typically more conserved than surface proteins and are often the target of CD8+ T cells. Therefore, we have characterized eight peptides derived from the internal SARS-CoV-2 nucleocapsid protein predicted to bind HLA-A∗02:01, the most common HLA molecule in the global population. We determined not all peptides could form a complex with HLA-A∗02:01, and the six crystal structures determined revealed that some peptides adopted a mobile conformation. We therefore provide a molecular understanding of SARS-CoV-2 CD8+ T cell epitopes. Furthermore, we show that there is limited pre-existing CD8+ T cell response toward these epitopes in unexposed individuals. Together, these data show that SARS-CoV-2 nucleocapsid might not contain potent epitopes restricted to HLA-A∗02:01.
RESUMO
The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus; however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Epitopos de Linfócito T/química , Antígeno HLA-A2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Linfócitos T CD8-Positivos/citologia , Cristalografia por Raios X , Citocinas/metabolismo , Epitopos/química , Antígeno HLA-A2/química , Humanos , Mutação , Peptídeos/química , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Ressonância de Plasmônio de Superfície , Linfócitos T Citotóxicos/imunologiaRESUMO
Histocompatibility Leukocyte Antigens, or HLAs, are one of the most polymorphic molecules in humans. This high degree of polymorphism endows HLA molecules with the ability to present a vast array of peptides, an essential trait for responding to ever-evolving pathogens. Unlike classical HLA molecules (HLA-Ia), some non-classical HLA-Ib molecules, including HLA-E, are almost monomorphic. Several studies show HLA-E can present self-peptides originating from the leader sequence of other HLA molecules, which signals to our immune system that the cell is healthy. Therefore, it was traditionally thought that the chief role of HLA-E in the body was in immune surveillance. However, there is emerging evidence that HLA-E is also able to present pathogen-derived peptides to the adaptive immune system, namely T cells, in a manner that is similar to classical HLA-Ia molecules. Here we describe the early findings of this less conventional role of HLA-E in the adaptive immune system and its importance for immunity.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade Adaptativa , Sequência de Aminoácidos , Apresentação de Antígeno/imunologia , Sítios de Ligação , Infecções por Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por HIV/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Modelos Moleculares , Polimorfismo Genético , Conformação Proteica , Infecções por Salmonella/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Tuberculose/imunologia , Antígenos HLA-ERESUMO
A member of the ribonucleaseâ A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced-fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity inâ vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell-binding sites, NMR studies indicate greater affinity for the cell-binding site than for the active site. Additionally, molecular dynamics simulations at 100â ns confirmed the stability of binding at the cell-binding site with the predicted protein-ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng inâ vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity.
Assuntos
Nucleosídeos de Pirimidina/química , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Simulação por Computador , Humanos , Simulação de Dinâmica Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Ressonância Magnética Nuclear Biomolecular , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. OBJECTIVE: The aim was to examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. METHOD: Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. RESULTS: All extracts exhibited significant in vitro inhibitory potency (IC50 values in the range of low µg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively.While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3Mthat acts synergistically with ellagic acid. CONCLUSION: Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements, our study provides a first positive step towards this process.