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1.
J Cell Sci ; 135(9)2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35393611

RESUMO

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (ß2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for ß2m.


Assuntos
Antígenos de Histocompatibilidade Classe I , Microglobulina beta-2 , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Ligação Proteica , Microglobulina beta-2/metabolismo
2.
J Biol Chem ; 298(2): 101542, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34968463

RESUMO

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included ß2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.


Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I , Complexo Principal de Histocompatibilidade , Antígenos de Histocompatibilidade Menor , ATPases do Tipo-P , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , ATPases do Tipo-P/imunologia
3.
J Cell Sci ; 134(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34085696

RESUMO

NKG2D (also known as KLRK1) is a crucial natural killer (NK) cell-activating receptor, and the murine cytomegalovirus (MCMV) employs multiple immunoevasins to avoid NKG2D-mediated activation. One of the MCMV immunoevasins, gp40 (m152), downregulates the cell surface NKG2D ligand RAE-1γ (also known as Raet1c) thus limiting NK cell activation. This study establishes the molecular mechanism by which gp40 retains RAE-1γ in the secretory pathway. Using flow cytometry and pulse-chase analysis, we demonstrate that gp40 retains RAE-1γ in the early secretory pathway, and that this effect depends on the binding of gp40 to a host protein, TMED10, a member of the p24 protein family. We also show that the TMED10-based retention mechanism can be saturated, and that gp40 has a backup mechanism as it masks RAE-1γ on the cell surface, blocking the interaction with the NKG2D receptor and thus NK cell activation.


Assuntos
Muromegalovirus , Animais , Ligantes , Proteínas de Membrana , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas Virais
4.
Ann Rheum Dis ; 82(7): 887-896, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36987655

RESUMO

The 'MHC-I (major histocompatibility complex class I)-opathy' concept describes a family of inflammatory conditions with overlapping clinical manifestations and a strong genetic link to the MHC-I antigen presentation pathway. Classical MHC-I-opathies such as spondyloarthritis, Behçet's disease, psoriasis and birdshot uveitis are widely recognised for their strong association with certain MHC-I alleles and gene variants of the antigen processing aminopeptidases ERAP1 and ERAP2 that implicates altered MHC-I peptide presentation to CD8+T cells in the pathogenesis. Progress in understanding the cause and treatment of these disorders is hampered by patient phenotypic heterogeneity and lack of systematic investigation of the MHC-I pathway.Here, we discuss new insights into the biology of MHC-I-opathies that strongly advocate for disease-overarching and integrated molecular and clinical investigation to decipher underlying disease mechanisms. Because this requires transformative multidisciplinary collaboration, we introduce the EULAR study group on MHC-I-opathies to unite clinical expertise in rheumatology, dermatology and ophthalmology, with fundamental and translational researchers from multiple disciplines such as immunology, genomics and proteomics, alongside patient partners. We prioritise standardisation of disease phenotypes and scientific nomenclature and propose interdisciplinary genetic and translational studies to exploit emerging therapeutic strategies to understand MHC-I-mediated disease mechanisms. These collaborative efforts are required to address outstanding questions in the etiopathogenesis of MHC-I-opathies towards improving patient treatment and prognostication.


Assuntos
Síndrome de Behçet , Espondilartrite , Uveíte , Humanos , Predisposição Genética para Doença , Síndrome de Behçet/genética , Antígenos de Histocompatibilidade Classe I/genética , Aminopeptidases/genética , Antígenos de Histocompatibilidade Menor/genética
5.
Proc Natl Acad Sci U S A ; 116(11): 5055-5060, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30808808

RESUMO

MHC-I epitope presentation to CD8+ T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Leucina/genética , Proteínas de Membrana Transportadoras/química , Camundongos Knockout , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína
6.
Nat Chem Biol ; 20(5): 549-550, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38580838
7.
Int J Mol Sci ; 21(23)2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33266306

RESUMO

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.


Assuntos
Catepsinas/biossíntese , Células Epiteliais da Tireoide/metabolismo , Tireotropina/metabolismo , Sequência de Aminoácidos , Biomarcadores , Catepsinas/química , Catepsinas/genética , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Imunofluorescência , Expressão Gênica , Genes Reporter , Glicosilação , Humanos , Lisossomos/metabolismo , Transporte Proteico , Glândula Tireoide/metabolismo
8.
J Cell Sci ; 129(1): 219-27, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26527401

RESUMO

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Muromegalovirus/metabolismo , Via Secretória , Proteínas Virais/metabolismo , Animais , Camundongos , Modelos Biológicos , Peptídeos/metabolismo , Ligação Proteica
9.
J Immunol ; 197(9): 3454-3463, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664280

RESUMO

Cross-presentation of phagocytosed Ags by MHC class I (MHC-I) molecules is thought to involve transport of cytosolic peptides into dendritic cell phagosomes, mediated by TAP transporters recruited from the endoplasmic reticulum. However, because pure and tightly sealed phagosomes are difficult to obtain, direct evidence for peptide transport into phagosomes has remained limited. Moreover, the parameters determining peptide uptake by, and survival in, phagosomes remain little characterized. In this study, we monitored peptide import into phagosomes by flow cytometry using two types of fluorescent reporter peptides, one of which directly bound to intraphagosomal beads. We observed that a peptide with high TAP affinity is imported into phagosomes in a TAP- and ATP-dependent manner, as expected. However, surprisingly, import of the OVA peptide SIINFEKL, a CD8+ T cell epitope frequently used to study cross-presentation, is ATP-dependent but substantially TAP-independent. The half-life of both reporter peptides is shortened by enhanced phagosome maturation triggered by TLR signaling. Conversely, formation of complexes with MHC-I molecules enhances peptide accumulation in phagosomes. Collectively, these results confirm that TAP can import peptides into phagosomes, but they suggest that some peptides, including the popular SIINFEKL, can enter phagosomes also via a second unknown energy-dependent mechanism. Therefore, the frequently reported TAP dependence of cross-presentation of phagocytosed OVA may principally reflect a requirement for recycling MHC-I molecules rather than SIINFEKL import into phagosomes via TAP.


Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/fisiologia , Retículo Endoplasmático/metabolismo , Fagossomos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antígenos/metabolismo , Células Cultivadas , Apresentação Cruzada , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/metabolismo , Peptídeos/metabolismo , Fagocitose
10.
Proc Natl Acad Sci U S A ; 112(1): 202-7, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25535340

RESUMO

Peptide ligand selection by MHC class I molecules, which occurs by iterative optimization, is the centerpiece of immunodominance in antiviral and antitumor immune responses. For its understanding, the molecular mechanisms of peptide binding and dissociation by class I molecules must be elucidated. To this end, we have investigated dipeptides that bind to the F pocket of class I molecules. We find that they accelerate the dissociation of prebound peptides of both low and high affinity, suggesting a mechanism of action for the peptide-exchange chaperone tapasin. Peptide exchange on class I molecules also has practical uses in epitope discovery and T-cell monitoring.


Assuntos
Biocatálise , Dipeptídeos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Sequência de Aminoácidos , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Multimerização Proteica
11.
Traffic ; 16(6): 591-603, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25753898

RESUMO

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi-step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER-Golgi intermediate compartment or the cis-Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H-2D(b) and H-2K(b) . We find that a novel pre-ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Imunológicos/metabolismo , Via Secretória , Animais , Calreticulina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Ligação Proteica , Dobramento de Proteína , Transporte Proteico
12.
Small ; 13(15)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28151581

RESUMO

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2Kb , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2Kb and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Animais , Membrana Celular/metabolismo , Sobrevivência Celular , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Ligação Proteica
13.
J Immunol ; 195(9): 4503-13, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26416272

RESUMO

The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I molecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes.


Assuntos
Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Polarização de Fluorescência , Antígeno HLA-B44/química , Antígeno HLA-B44/genética , Antígeno HLA-B44/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/metabolismo , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Terciária de Proteína , Termodinâmica
14.
Anesth Analg ; 125(4): 1246-1252, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28368939

RESUMO

BACKGROUND: Traditionally, mechanical ventilation is achieved via active lung inflation during inspiration and passive lung emptying during expiration. By contrast, the novel FLEX (FLow-controlled EXpiration) ventilator mode actively decreases the rate of lung emptying. We investigated whether FLEX can be used during intraoperative mechanical ventilation of lung-healthy patients. METHODS: In 30 adult patients scheduled for neurosurgical procedures, we studied respiratory system mechanics, regional ventilation, oxygenation, and hemodynamics during ventilation with and without FLEX at positive end-expiratory pressure (PEEP) of 5 and 7 cm H2O. The FLEX system was integrated into the expiratory limb and modified the expiratory flow profile by continuously changing expiratory resistance according to a computer-controlled algorithm. RESULTS: Mean airway pressure increased with PEEP by 1.9 cm H2O and with FLEX by 1 cm H2O (all P < .001). The expiratory peak flow was 42% lower with FLEX than without FLEX (P < .001). FLEX caused significant shifts in aeration from ventral to the dorsal lung regions. Respiratory mechanics, end-tidal carbon dioxide partial pressure, oxygenation, and hemodynamics were independent from FLEX and PEEP. We observed no critical incidents or FLEX malfunctions in any measurement that would have required an intervention or termination of the FLEX mode. CONCLUSIONS: FLEX can be used in lung-healthy patients who are mechanically ventilated during general anesthesia. FLEX improves the homogeneous distribution of ventilation in the lungs.


Assuntos
Hemodinâmica/fisiologia , Pulmão/fisiologia , Respiração Artificial/métodos , Mecânica Respiratória/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Cross-Over , Expiração/fisiologia , Feminino , Humanos , Inalação/fisiologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Respiração com Pressão Positiva/métodos , Adulto Jovem
15.
Traffic ; 15(5): 531-45, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24479619

RESUMO

Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)-coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII-coat-forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that - owing to its FK506-binding protein domains - can be oligomerized in isolated membranes by addition of a small-molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization-dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Citosol/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Multimerização Proteica/fisiologia , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Ligação Proteica/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismo , Leveduras/metabolismo , Leveduras/fisiologia
16.
J Cell Sci ; 127(Pt 13): 2885-97, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24806963

RESUMO

The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K(b)-Y84C, in which two α-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. K(b)-Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (ß2m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and ß2m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by ß2m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that ß2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Células 3T3 , Animais , Apresentação de Antígeno , Endocitose , Epitopos , Antígenos H-2/química , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Células HeLa , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Estrutura Secundária de Proteína , Linfócitos T/imunologia , Linfócitos T/metabolismo
17.
Eur J Immunol ; 45(4): 1248-57, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25615938

RESUMO

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract.


Assuntos
Antígeno HLA-B27/química , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/metabolismo , Espondilite Anquilosante/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Antígeno HLA-B27/genética , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Análise de Sequência de DNA , Espondilite Anquilosante/genética
18.
FASEB J ; 29(7): 2780-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25782992

RESUMO

Major histocompatibility complex class I proteins, which present antigenic peptides to cytotoxic T lymphocytes at the surface of all nucleated cells, are endocytosed and destroyed rapidly once their peptide ligand has dissociated. The molecular mechanism of this cellular quality control process, which prevents rebinding of exogenous peptides and thus erroneous immune responses, is unknown. To identify the nature of the decisive step in endocytic sorting of class I molecules and its location, we have followed the removal of optimally and suboptimally peptide-loaded murine H-2K(b) class I proteins from the cell surface. We find that the binding of their light chain, ß2-microglobulin (ß2m), protects them from endocytic destruction. Thus, the extended survival of suboptimally loaded K(b) molecules at 25°C is attributed to decreased dissociation of ß2m. Because all forms of K(b) are constantly internalized but little ß2m-receptive heavy chain is present at the cell surface, it is likely that ß2m dissociation and recognition of the heavy chain for lysosomal degradation take place in an endocytic compartment.


Assuntos
Antígenos H-2/metabolismo , Microglobulina beta-2/metabolismo , Animais , Apresentação de Antígeno , Compartimento Celular , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Endocitose/imunologia , Camundongos , Modelos Imunológicos , Ligação Proteica , Estrutura Quaternária de Proteína , Proteólise , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Temperatura , Microglobulina beta-2/química
19.
J Immunol ; 192(11): 4957-66, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24778449

RESUMO

Peptide ligands presented by MHC class I (MHC-I) molecules are produced by degradation of cytosolic and nuclear, but also endoplasmic reticulum (ER)-resident, proteins by the proteasome. However, Ag processing of ER proteins remains little characterized. Studying processing and presentation of proinsulin, which plays a pivotal role in autoimmune diabetes, we found that targeting to the ER has profound effects not only on how proinsulin is degraded, but also on regulation of its cellular levels. While proteasome inhibition inhibited degradation and presentation of cytosolic proinsulin, as expected, it reduced the abundance of ER-targeted proinsulin. This targeting and protein modifications modifying protein half-life also had profound effects on MHC-I presentation and proteolytic processing of proinsulin. Thus, presentation of stable luminal forms was inefficient but enhanced by proteasome inhibition, whereas that of unstable luminal forms and of a cytosolic form were more efficient and compromised by proteasome inhibitors. Distinct stability of peptide MHC complexes produced from cytosolic and luminal proinsulin suggests that different proteolytic activities process the two Ag forms. Thus, both structural features and subcellular targeting of Ags can have strong effects on the processing pathways engaged by MHC-I-restricted Ags, and on the efficiency and regulation of their presentation.


Assuntos
Apresentação de Antígeno , Retículo Endoplasmático/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proinsulina/imunologia , Proteólise , Retículo Endoplasmático/genética , Regulação da Expressão Gênica/genética , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos/genética , Peptídeos/imunologia , Proinsulina/genética
20.
Proc Natl Acad Sci U S A ; 110(38): 15383-8, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24003162

RESUMO

MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2K(b) into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-2K(b) and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide.


Assuntos
Aminoácidos/metabolismo , Dipeptídeos/metabolismo , Antígeno HLA-A2/metabolismo , Conformação Proteica , Dipeptídeos/genética , Retículo Endoplasmático/metabolismo , Escherichia coli , Citometria de Fluxo , Polarização de Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Ligação Proteica , Dobramento de Proteína
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