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1.
Cell ; 164(1-2): 128-140, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26771488

RESUMO

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.


Assuntos
Integrinas/metabolismo , Macrófagos/imunologia , Fagocitose , Actinas/metabolismo , Animais , Humanos , Antígenos Comuns de Leucócito/metabolismo , Macrófagos/citologia , Camundongos , Podossomos/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Receptores de IgG/metabolismo
2.
EMBO J ; 43(1): 132-150, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38177315

RESUMO

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.


Assuntos
Antígenos , Linfócitos T , Ligantes , Receptores de Antígenos de Linfócitos T/metabolismo , Ativação Linfocitária
3.
EMBO J ; 42(7): e111841, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36484367

RESUMO

T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo Principal de Histocompatibilidade , Peptídeos , Simulação de Dinâmica Molecular , Ligação Proteica
4.
Proc Natl Acad Sci U S A ; 120(2): e2216352120, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36598945

RESUMO

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a > 100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only < 5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3ε subunit (also known as a TRuC) and fully restored by exchanging the TCRαß variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.


Assuntos
Receptores de Antígenos Quiméricos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Antígeno-1 Associado à Função Linfocitária , Ativação Linfocitária , Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD28/metabolismo
5.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35197288

RESUMO

Protein-protein binding domains are critical in signaling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins has tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signaling, which is a critical requirement of noise-filtering mechanisms such as kinetic proofreading. Here, we use modeling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases. Using surface plasmon resonance, we show that the phosphatase CD45 can accelerate the unbinding rate of zeta chain-associated protein kinase 70 (ZAP70), a tandem SH2 domain-containing kinase, from biphosphorylated peptides from the T cell receptor (TCR). An important functional prediction of accelerated unbinding is that the intracellular ZAP70-TCR half-life in T cells will not be fixed but rather, dependent on the extracellular TCR-antigen half-life, and we show that this is the case in both cell lines and primary T cells. The work highlights that tandem SH2 domains can break the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discrimination mediated by kinetic proofreading.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Regulação Alostérica , Meia-Vida , Humanos , Cinética , Fosforilação , Ligação Proteica
6.
PLoS Comput Biol ; 18(3): e1009922, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35235558

RESUMO

SARS-CoV-2 Spike (Spike) binds to human angiotensin-converting enzyme 2 (ACE2) and the strength of this interaction could influence parameters relating to virulence. To explore whether population variants in ACE2 influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities and kinetics for Spike receptor binding domain through surface plasmon resonance (SPR) at 37°C. We discovered variants that reduce and enhance binding, including three ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = -1.33 ± 0.15 kcal mol-1 and p.Gly352Val, predicted ΔΔG = -1.17 kcal mol-1) or abolished (p.Asp355Asn) binding. We also identified two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol-1) is predominant in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol-1) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. We compared ACE2 variant affinities to published SARS-CoV-2 pseudotype infectivity data and confirmed that ACE2 variants with reduced affinity for Spike can protect cells from infection. The effect of variants with enhanced Spike affinity remains unclear, but we propose a mechanism whereby these alleles could cause greater viral spreading across tissues and cell types, as is consistent with emerging understanding regarding the interplay between receptor affinity and cell-surface abundance. Finally, we compared mCSM-PPI2 ΔΔG predictions against our SPR data to assess the utility of predictions in this system. We found that predictions of decreased binding were well-correlated with experiment and could be improved by calibration, but disappointingly, predictions of highly enhanced binding were unreliable. Recalibrated predictions for all possible ACE2 missense variants at the Spike interface were calculated and used to estimate the overall burden of ACE2 variants on Covid-19.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Mutação de Sentido Incorreto , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Predisposição Genética para Doença , Humanos , Ligação Proteica
7.
Br J Cancer ; 126(12): 1663-1675, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35228677

RESUMO

The binding of T cell immune checkpoint proteins programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to their ligands allows immune evasion by tumours. The development of therapeutic antibodies, termed checkpoint inhibitors, that bind these molecules or their ligands, has provided a means to release this brake on the host anti-tumour immune response. However, these drugs are costly, are associated with potentially severe side effects, and only benefit a small subset of patients. It is therefore important to identify biomarkers that discriminate between responders and non-responders. This review discusses the determinants for a successful response to antibodies that bind PD-1 or its ligand PD-L1, dividing them into markers found in the tumour biopsy and those in non-tumour samples. It provides an update on the established predictive biomarkers (tumour PD-L1 expression, tumour mismatch repair deficiency and tumour mutational burden) and assesses the evidence for new potential biomarkers.


Assuntos
Antígeno B7-H1 , Neoplasias Colorretais , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Humanos , Ligantes , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia
8.
PLoS Biol ; 17(12): e3000549, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815943

RESUMO

Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor-cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.


Assuntos
Antígenos de Superfície/fisiologia , Bioensaio/métodos , Comunicação Celular/imunologia , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Células THP-1
9.
Immunity ; 34(1): 1-3, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21272780

RESUMO

In this issue of Immunity, Jiang et al. (2011) provide evidence that the CD8 coreceptor is recruited to the T cell receptor (TCR) complex after initial TCR triggering where it stabilizes the TCR-peptide-major histocompatibility complex interaction.

10.
PLoS Comput Biol ; 15(9): e1007338, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31498801

RESUMO

T cells use their T-cell receptors (TCRs) to scan other cells for antigenic peptides presented by MHC molecules (pMHC). If a TCR encounters a pMHC, it can trigger a signalling pathway that could lead to the activation of the T cell and the initiation of an immune response. It is currently not clear how the binding of pMHC to the TCR initiates signalling within the T cell. One hypothesis is that conformational changes in the TCR lead to further downstream signalling. Here we investigate four different TCRs in their free state as well as in their pMHC bound state using large scale molecular simulations totalling 26 000 ns. We find that the dynamical features within TCRs differ significantly between unbound TCR and TCR/pMHC simulations. However, apart from expected results such as reduced solvent accessibility and flexibility of the interface residues, these features are not conserved among different TCR types. The presence of a pMHC alone is not sufficient to cause cross-TCR-conserved dynamical features within a TCR. Our results argue against models of TCR triggering involving conserved allosteric conformational changes.


Assuntos
Antígenos de Histocompatibilidade , Receptores de Antígenos de Linfócitos T , Biologia Computacional , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
12.
Bioinformatics ; 34(11): 1941-1943, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29329361

RESUMO

Motivation: Hydrogen bonds (H-bonds) play an essential role for many molecular interactions but are also often transient, making visualising them in a flexible system challenging. Results: We provide pyHVis3D which allows for an easy to interpret 3D visualisation of H-bonds resulting from molecular simulations. We demonstrate the power of pyHVis3D by using it to explain the changes in experimentally measured binding affinities for three T-cell receptor/peptide/MHC complexes and mutants of each of these complexes. Availability and implementation: pyHVis3D can be downloaded for free from http://opig.stats.ox.ac.uk/resources. Contact: science.bernhard.knapp@gmail.com. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Biologia Computacional/métodos , Simulação de Dinâmica Molecular , Receptores de Antígenos de Linfócitos T/metabolismo , Software , Algoritmos , Humanos , Ligação de Hidrogênio , Mutação , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética
13.
Immunity ; 32(2): 163-74, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20137987

RESUMO

T cell receptor (TCR) binding to diverse peptide-major histocompatibility complex (pMHC) ligands results in various degrees of T cell activation. Here we analyze which binding properties of the TCR-pMHC interaction are responsible for this variation in pMHC activation potency. We have analyzed activation of the 1G4 cytotoxic T lymphocyte clone by cognate pMHC variants and performed thorough correlation analysis of T cell activation with 1G4 TCR-pMHC binding properties measured in solution. We found that both the on rate (k(on)) and off rate (k(off)) contribute to activation potency. Based on our results, we propose a model in which rapid TCR rebinding to the same pMHC after chemical dissociation increases the effective half-life or "confinement time" of a TCR-pMHC interaction. This confinement time model clarifies the role of k(on) in T cell activation and reconciles apparently contradictory reports on the role of TCR-pMHC binding kinetics and affinity in T cell activation.


Assuntos
Antígeno HLA-A2/metabolismo , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Clonais , Citotoxicidade Imunológica , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Modelos Imunológicos , Proteínas de Neoplasias/química , Fragmentos de Peptídeos/química , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Ressonância de Plasmônio de Superfície , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Fatores de Tempo , Transfecção
14.
Immunity ; 30(3): 348-57, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19303388

RESUMO

Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen. Structural analysis demonstrates this crossreactivity is due to structural mimicry of a binding hotspot shared by self and microbial antigens, rather than to degenerate TCR recognition. Biophysical studies reveal that the autoreactive TCR binding affinity is markedly lower for the microbial (mimicry) peptide than for the autoantigenic peptide. Thus, these data suggest a possible explanation for the difficulty in incriminating individual infections in the development of MS.


Assuntos
Doenças Autoimunes/imunologia , Proteínas de Bactérias/imunologia , Mimetismo Molecular/imunologia , Peptídeos/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Cerebelo/patologia , Reações Cruzadas/imunologia , Drosophila , Escherichia coli/imunologia , Antígenos HLA-D/metabolismo , Antígeno HLA-DR2/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Esclerose Múltipla/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Medula Espinal/patologia , Linfócitos T/fisiologia
15.
J Biol Chem ; 291(48): 25004-25018, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27733683

RESUMO

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.


Assuntos
Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Ácido Hialurônico/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Humanos , Ácido Hialurônico/genética , Oxirredução , Proteínas de Transporte Vesicular/genética
17.
Trends Immunol ; 35(4): 153-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24636916

RESUMO

T cells have to detect rare high-affinity 'foreign' peptide MHC (pMHC) ligands among abundant low-affinity 'self'-peptide MHC ligands. It remains unclear how this remarkable discrimination is achieved. Kinetic proofreading mechanisms can provide the required specificity but only at the expense of much reduced sensitivity. A number of recent observations suggest that pMHC engagement of T cell receptors (TCRs) induces changes such as clustering and/or conformational alterations that enhance subsequent rebinding. We show that inclusion of induced rebinding to the same pMHC in kinetic proofreading models enhances the sensitivity of TCR recognition while retaining specificity. Moreover, induced rebinding is able to reproduce the striking, and hitherto unexplained, 2D membrane-binding properties recently reported for the TCR.


Assuntos
Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Humanos , Ligação Proteica/imunologia , Tolerância a Antígenos Próprios/imunologia
18.
J Immunol ; 195(11): 5432-9, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26500347

RESUMO

Optimal T cell activation typically requires engagement of both the TCR and costimulatory receptors, such as CD28. Engagement of CD28 leads to tyrosine phosphorylation of its cytoplasmic region and recruitment of cytoplasmic signaling proteins. Although the exact mechanism of CD28 signal transduction is unknown, CD28 triggering has similarities to the TCR, which was proposed to use the kinetic-segregation (KS) mechanism. The KS model postulates that, when small receptors engage their ligands within areas of close (∼15 nm) contact in the T cell/APC interface, this facilitates phosphorylation by segregating the engaged receptor/ligand complex from receptor protein tyrosine phosphatases with large ectodomains, such as CD45. To test this hypothesis, we examined the effect of elongating the extracellular region of the CD28 ligand, CD80, on its ability to costimulate IL-2 production by primary T cells. CD80 elongation reduced its costimulatory effect without abrogating CD28 binding. Confocal microscopy revealed that elongated CD80 molecules were less well segregated from CD45 at the T cell/APC interface. T cells expressing CD28 harboring a key tyrosine-170 mutation were less sensitive to CD80 elongation. In summary, the effectiveness of CD28 costimulation is inversely proportional to the dimensions of the CD28-CD80 complex. Small CD28-CD80 complex dimensions are required for optimal costimulation by segregation from large inhibitory tyrosine phosphatases. These results demonstrate the importance of ligand dimensions for optimal costimulation of IL-2 production by T cells and suggest that the KS mechanism contributes to CD28 signaling.


Assuntos
Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/biossíntese , Animais , Antígeno B7-1/genética , Antígenos CD28/genética , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Fosforilação/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia
19.
Biophys J ; 110(8): 1896-1906, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27119648

RESUMO

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células HEK293 , Humanos , Cinética , Camundongos , Fosforilação , Receptores de Antígenos de Linfócitos T/química
20.
N Engl J Med ; 369(21): 2012-2020, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24195502

RESUMO

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.


Assuntos
Mutação em Linhagem Germinativa , Hiperprolactinemia/genética , Receptores da Prolactina/genética , Adulto , Feminino , Humanos , Janus Quinase 2/metabolismo , Masculino , Linhagem , Conformação Proteica , Receptores da Prolactina/química , Fator de Transcrição STAT5/fisiologia , Análise de Sequência de DNA , Transdução de Sinais/fisiologia
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