RESUMEN

The last decade has seen significant progress in understanding how the genome is organized spatially within interphase nuclei. Recent analyses have confirmed earlier molecular cytogenetic studies on chromosome positioning within interphase nuclei and provided new information about the topologically associated domains (TADs). Examining the nuances of how genomes are organized within interphase nuclei will provide information fundamental to understanding gene regulation and expression in health and disease. Indeed, the radial spatial positioning of individual gene loci within nuclei has been associated with up- and down-regulation of specific genes, and disruption of normal genome organization within nuclei will result in compromised cellular health. In cancer cells, where reorganization of the nuclear architecture may occur in the presence of chromosomal rearrangements such as translocations, inversions, or deletions, gene repositioning can change their expression. To date, very few studies have focused on radial gene positioning and the correlation to gene expression in cancers. Further investigations would improve our understanding of the biological mechanisms at the basis of cancer and, in particular, in leukemia initiation and progression, especially in those cases where the molecular consequences of chromosomal rearrangements are still unclear. In this review, we summarize the main milestones in the field of genome organization in the nucleus and the alterations to this organization that can lead to cancer diseases.

RESUMEN

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, premature ageing syndrome in children. HGPS is normally caused by a mutation in the LMNA gene, encoding nuclear lamin A. The classical mutation in HGPS leads to the production of a toxic truncated version of lamin A, progerin, which retains a farnesyl group. Farnesyltransferase inhibitors (FTI), pravastatin and zoledronic acid have been used in clinical trials to target the mevalonate pathway in HGPS patients to inhibit farnesylation of progerin, in order to reduce its toxicity. Some other compounds that have been suggested as treatments include rapamycin, IGF1 and N-acetyl cysteine (NAC). We have analysed the distribution of prelamin A, lamin A, lamin A/C, progerin, lamin B1 and B2 in nuclei of HGPS cells before and after treatments with these drugs, an FTI and a geranylgeranyltransferase inhibitor (GGTI) and FTI with pravastatin and zoledronic acid in combination. Confirming other studies prelamin A, lamin A, progerin and lamin B2 staining was different between control and HGPS fibroblasts. The drugs that reduced progerin staining were FTI, pravastatin, zoledronic acid and rapamycin. However, drugs affecting the mevalonate pathway increased prelamin A, with only FTI reducing internal prelamin A foci. The distribution of lamin A in HGPS cells was improved with treatments of FTI, pravastatin and FTI + GGTI. All treatments reduced the number of cells displaying internal speckles of lamin A/C and lamin B2. Drugs targeting the mevalonate pathway worked best for progerin reduction, with zoledronic acid removing internal progerin speckles. Rapamycin and NAC, which impact on the MTOR pathway, both reduced both pools of progerin without increasing prelamin A in HGPS cell nuclei.

Asunto(s)

Lamina Tipo A/metabolismo , Ácido Mevalónico/metabolismo , Progeria/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Progeria/patología

RESUMEN

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

Asunto(s)

Butiratos/farmacología , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , PPAR alfa/agonistas , Compuestos de Fenilurea/farmacología , Butiratos/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Ligandos , Modelos Moleculares , Mutación , Compuestos de Fenilurea/metabolismo , Conformación Proteica/efectos de los fármacos

RESUMEN

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.

Asunto(s)

Daño del ADN/efectos de los fármacos , Difosfonatos/uso terapéutico , Farnesiltransferasa/antagonistas & inhibidores , Genoma Humano/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Progeria/tratamiento farmacológico , Sirolimus/uso terapéutico , Línea Celular , Ensayo Cometa , Difosfonatos/farmacología , Quimioterapia Combinada , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Lamina Tipo A/genética , Laminas/genética , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Mutación , Progeria/genética , Progeria/metabolismo , Procesamiento Proteico-Postraduccional , Sirolimus/farmacología

RESUMEN

αß T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8+ T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8+ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9+ TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9+ TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14+ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.

Asunto(s)

Linfocitos T CD8-positivos/metabolismo , Metabolismo Energético , Antígenos de Histocompatibilidad Clase I/metabolismo , Modelos Moleculares , Receptores de Antígenos de Linfocitos T alfa-beta/agonistas , Secuencia de Aminoácidos , Sitios de Unión , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Células Clonales , Secuencia Conservada , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos de Linfocito T , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Antígenos HLA-E

RESUMEN

The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH.

Asunto(s)

Núcleo Celular/metabolismo , Genoma , Hibridación Fluorescente in Situ , Membrana Nuclear/metabolismo , Biomarcadores , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Sondas de ADN , Antígeno Ki-67/metabolismo , Microscopía Fluorescente

RESUMEN

The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.

Asunto(s)

Antígenos HLA-B/química , Antígenos HLA-G/química , Receptores KIR2DL4/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Mariposas Nocturnas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores KIR2DL4/genética , Receptores KIR2DL4/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia

RESUMEN

The primary function of the monomorphic MHC class Ib molecule Qa-1(b) is to present peptides derived from the leader sequences of other MHC class I molecules for recognition by the CD94-NKG2 receptors expressed by NK and T cells. Whereas the mode of peptide presentation by its ortholog HLA-E, and subsequent recognition by CD94-NKG2A, is known, the molecular basis of Qa-1(b) function is unclear. We have assessed the interaction between Qa-1(b) and CD94-NKG2A and shown that they interact with an affinity of 17 µM. Furthermore, we have determined the structure of Qa-1(b) bound to the leader sequence peptide, Qdm (AMAPRTLLL), to a resolution of 1.9 Å and compared it with that of HLA-E. The crystal structure provided a basis for understanding the restricted peptide repertoire of Qa-1(b). Whereas the Qa-1(b-AMAPRTLLL) complex was similar to that of HLA-E, significant sequence and structural differences were observed between the respective Ag-binding clefts. However, the conformation of the Qdm peptide bound by Qa-1(b) was very similar to that of peptide bound to HLA-E. Although a number of conserved innate receptors can recognize heterologous ligands from other species, the structural differences between Qa-1(b) and HLA-E manifested in CD94-NKG2A ligand recognition being species specific despite similarities in peptide sequence and conformation. Collectively, our data illustrate the structural homology between Qa-1(b) and HLA-E and provide a structural basis for understanding peptide repertoire selection and the specificity of the interaction of Qa-1(b) with CD94-NKG2 receptors.

Asunto(s)

Presentación de Antígeno/fisiología , Antígenos de Histocompatibilidad Clase I/química , Subfamília C de Receptores Similares a Lectina de Células NK/química , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Péptidos/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Ratones , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Péptidos/genética , Péptidos/inmunología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad de la Especie , Homología Estructural de Proteína , Relación Estructura-Actividad , Antígenos HLA-E

RESUMEN

Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards ß2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an 'innate HLA sensor' domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species.

Asunto(s)

Antígenos HLA-B/química , Antígenos HLA-B/inmunología , Receptores KIR3DL1/química , Receptores KIR3DL1/inmunología , Secuencia de Aminoácidos , Sitios de Unión/genética , Antígenos HLA-B/genética , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Polimorfismo Genético/genética , Estructura Terciaria de Proteína , Receptores KIR3DL1/genética , Relación Estructura-Actividad , Microglobulina beta-2/química , Microglobulina beta-2/inmunología

RESUMEN

alphabeta T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

Asunto(s)

Inmunidad Adaptativa , Complejo Mayor de Histocompatibilidad , Receptores de Antígenos de Linfocitos T/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Bases de Datos Genéticas , Humanos , Activación de Linfocitos , Modelos Moleculares , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética

RESUMEN

Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of "independent pegs" that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of alphabeta-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many self- and viral peptides show extensive and direct interresidue interactions, an unexpected finding that has led us to the idea of "constrained" peptides. Mutational analysis of two constrained peptides (the HLA B44 restricted self-peptide (B44DPalpha-EEFGRAFSF) and an H2-D(b) restricted influenza peptide (D(b)PA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLENFRAYV --> SSLENARAYV) within the constrained PA peptide resulted in a diminished cytotoxic T lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between individual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, whereas the perturbation of such constraints can lead to a previously unappreciated mechanism of viral escape.

Asunto(s)

Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Presentación de Antígeno , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Femenino , Antígenos H-2/química , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Antígeno HLA-B44 , Antígeno de Histocompatibilidad H-2D , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Conformación Proteica , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunología

RESUMEN

The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse alphabeta T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-G(KGPPAALTL) was very similar to that of the HLA-G(RIIPRHLQL) structure. However, the structure of HLA-G(KLPQAFYIL) not only differed in the conformation of the bound peptide but also caused a small shift in the alpha2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.

Asunto(s)

Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Cristalografía por Rayos X , Antígenos HLA-G , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Temperatura

RESUMEN

Ligation of the alphabeta T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha). The apparent affinity constant of this A-B loop movement mirrored that of alphabeta TCR-pMHC ligation and was observed in two alphabeta TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Calpha domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering.

Asunto(s)

Antígenos/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Linfocitos T/inmunología , Animales , Antígenos/inmunología , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Mutación/genética , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología

RESUMEN

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405(EENLLDFVRF) complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

Asunto(s)

Epítopos de Linfocito T/inmunología , Antígenos HLA-B/genética , Polimorfismo de Nucleótido Simple , Receptores de Antígenos de Linfocitos T/inmunología , Sustitución de Aminoácidos , Sitios de Unión , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Epítopos de Linfocito T/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/metabolismo , Antígeno HLA-B44 , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas Recombinantes/metabolismo , Linfocitos T Citotóxicos/inmunología

RESUMEN

During selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire.

Asunto(s)

Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Autotolerancia/inmunología , Secuencia de Aminoácidos , Antígenos Virales/química , Antígenos Virales/inmunología , Cristalografía por Rayos X , Antígeno HLA-B8/química , Antígeno HLA-B8/inmunología , Herpesvirus Humano 4/química , Herpesvirus Humano 4/inmunología , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Estructura Cuaternaria de Proteína , Homología Estructural de Proteína , Resonancia por Plasmón de Superficie

RESUMEN

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.

Asunto(s)

Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Epítopos Inmunodominantes/inmunología , Melanoma/inmunología , Proteínas de la Membrana/inmunología , Fragmentos de Péptidos/inmunología , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Antígenos HLA-B/inmunología , Antígeno HLA-B7 , Humanos , Activación de Linfocitos , Melanoma/terapia , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica

RESUMEN

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

Asunto(s)

Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos HLA-B/inmunología , Imitación Molecular/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Línea Celular , Antígeno HLA-B8 , Humanos , Células Jurkat , Activación de Linfocitos/inmunología , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Transfección

RESUMEN

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

Asunto(s)

Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Antígenos HLA/química , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Complejos Multiproteicos , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK , Unión Proteica , Señales de Clasificación de Proteína/genética , Subunidades de Proteína , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores de Células Asesinas Naturales , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Antígenos HLA-E

RESUMEN

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I molecule that binds peptides derived from the leader sequences of other HLA class I molecules. Natural killer cell recognition of these HLA-E molecules, via the CD94-NKG2 natural killer family, represents a central innate mechanism for monitoring major histocompatibility complex expression levels within a cell. The leader sequence-derived peptides bound to HLA-E exhibit very limited polymorphism, yet subtle differences affect the recognition of HLA-E by the CD94-NKG2 receptors. To better understand the basis for this peptide-specific recognition, we determined the structure of HLA-E in complex with two leader peptides, namely, HLA-Cw*07 (VMAPRALLL), which is poorly recognised by CD94-NKG2 receptors, and HLA-G*01 (VMAPRTLFL), a high-affinity ligand of CD94-NKG2 receptors. A comparison of these structures, both of which were determined to 2.5-A resolution, revealed that allotypic variations in the bound leader sequences do not result in conformational changes in the HLA-E heavy chain, although subtle changes in the conformation of the peptide within the binding groove of HLA-E were evident. Accordingly, our data indicate that the CD94-NKG2 receptors interact with HLA-E in a manner that maximises the ability of the receptors to discriminate between subtle changes in both the sequence and conformation of peptides bound to HLA-E.

Asunto(s)

Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Receptores Inmunológicos/inmunología , Antígenos HLA/química , Antígenos de Histocompatibilidad Clase I/química , Humanos , Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Conformación Proteica , Receptores Inmunológicos/metabolismo , Antígenos HLA-E

RESUMEN

The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

Asunto(s)

Antígenos HLA/química , Antígenos de Histocompatibilidad Clase I/química , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Dimerización , Humanos , Datos de Secuencia Molecular , Mutagénesis , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamília D de Receptores Similares a Lectina de las Células NK/fisiología , Subunidades de Proteína , Receptores Inmunológicos/fisiología , Receptores de Células Asesinas Naturales , Antígenos HLA-E