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 fármacosRESUMEN
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/patologíaRESUMEN
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-ERESUMEN
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 SuperficieRESUMEN
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 , TransfecciónRESUMEN
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/inmunologíaRESUMEN
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/farmacologíaRESUMEN
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/inmunologíaRESUMEN
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 SecuenciaRESUMEN
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-ERESUMEN
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/genéticaRESUMEN
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/inmunologíaRESUMEN
Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD1/inmunología , Glucolípidos/inmunología , Células Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno/genética , Antígenos CD1/genética , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T/genética , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Humanos , Ratones , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína/fisiología , Especificidad de la Especie , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
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
HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.
Asunto(s)
Antígenos HLA-B/genética , Linfocitos T/inmunología , Alelos , Línea Celular , Cristalografía por Rayos X , Citocinas/sangre , Frecuencia de los Genes , Antígenos HLA-B/química , Antígeno HLA-B44 , Humanos , Prueba de Cultivo Mixto de Linfocitos , Modelos Moleculares , Estructura Secundaria de Proteína , Caracteres Sexuales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The interaction between the alphabeta T cell receptor (TCR) and the peptide bound to the major histocompatibility complex class I molecule (pMHC-I) constitutes a central interaction in adaptive immunity. How these receptors interact with such low affinity while maintaining exquisite specificity for peptide antigen and host MHC (MHC-I restriction) remains a challenge to be explained by structural immunologists. Moreover, how this extracellular interaction is transmitted as an intracellular signal via the CD3 complex remains unresolved. Nevertheless, several structures of TCRs, non-liganded and ligated to a defined pMHC-I, combined with detailed biophysical analyses, have provided insight of the structural basis of MHC-I restriction. In addition, structures of isolated CD3 components have enabled T cell signalling mechanisms to be postulated. Recent findings in this area, which include seven distinct TCR/pMHC-I complexes, have fundamental implications in adaptive immunity as well as therapeutic applications to modulate the adaptive immune response.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Animales , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Modelos Inmunológicos , Modelos Moleculares , Complejos Multiproteicos , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
Human leukocyte antigen-G (HLA-G) is a class Ib major histocompatibility complex (MHC) molecule that is specifically expressed in immune-privileged tissues. The overall structure of HLA-G resembles other class I MHC molecules, in which a heavy chain comprised of three domains is noncovalently associated with beta(2)microglobulin (beta(2)m). A nine-residue self-peptide is bound within a cleft formed by two alpha-helices and a beta-sheet floor. An extensive network of contacts is formed between the peptide and the binding cleft, leading to a constrained mode of binding reminiscent of that observed in HLA-E. The alpha3 domain of HLA-G, the putative binding site for leukocyte immunoglobulinlike receptor-1 (LIR-1) and -2, is structurally distinct from class Ia MHC molecules, providing a basis for the observed differences in affinity for these ligands. In addition, a disulfide-bonded dimer adopts an oblique conformation, providing the possibility of a 1:2 (HLA-G dimer:receptor) complex stoichiometry. The relative orientation of the HLA-G protomers in the dimer structure suggests that it is unlikely that dimerization is involved in killer immunoglobulinlike receptor 2DL4 (KIR2DL4) binding.
Asunto(s)
Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Receptores Inmunológicos/metabolismo , Dimerización , Antígenos HLA-G , Humanos , Ligandos , Unión Proteica/fisiología , Receptores KIR2DL5RESUMEN
The organization of the genome within interphase nuclei, and how it interacts with nuclear structures is important for the regulation of nuclear functions. Many of the studies researching the importance of genome organization and nuclear structure are performed in young, proliferating, and often transformed cells. These studies do not reveal anything about the nucleus or genome in nonproliferating cells, which may be relevant for the regulation of both proliferation and replicative senescence. Here, we provide an overview of what is known about the genome and nuclear structure in senescent cells. We review the evidence that nuclear structures, such as the nuclear lamina, nucleoli, the nuclear matrix, nuclear bodies (such as promyelocytic leukemia bodies), and nuclear morphology all become altered within growth-arrested or senescent cells. Specific alterations to the genome in senescent cells, as compared to young proliferating cells, are described, including aneuploidy, chromatin modifications, chromosome positioning, relocation of heterochromatin, and changes to telomeres.
Asunto(s)
Núcleo Celular/metabolismo , Senescencia Celular , Envejecimiento , Animales , Nucléolo Celular/metabolismo , Proliferación Celular , Cromosomas/metabolismo , Cromosomas/ultraestructura , Modelos Animales de Enfermedad , Genoma , Humanos , Leucemia Promielocítica Aguda/metabolismo , Ratones , Modelos Biológicos , Matriz Nuclear/metabolismo , Telómero/ultraestructuraRESUMEN
HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal-maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 A resolution, belonged to space group P3(2)2(1), with unit-cell parameters a = b = 77.15, c = 151.72 A.
Asunto(s)
Cobalto/química , Antígenos HLA/química , Antígenos HLA/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Sustitución de Aminoácidos/genética , Cationes Bivalentes/química , Línea Celular Tumoral , Cisteína/genética , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Serina/genética , SolubilidadRESUMEN
Despite a potential repertoire of >10(15) alphabeta T cell receptors (TcR), the HLA B8-restricted cytolytic T cell response to a latent antigen of Epstein-Barr virus (EBV) is strikingly limited in the TcR sequences that are selected. Even in unrelated individuals this response is dominated by a single highly restricted TcR clonotype that selects identical combinations of hypervariable Valpha, Vbeta, D, J, and N region genes. We have determined the 1.5 A crystal structure of this "public" TcR, revealing that five of the six hypervariable loops adopt novel conformations providing a unique combining site that contains a deep pocket predicted to overlay the HLA B8-peptide complex. The findings suggest a structural basis for the immunodominance of this clonotype in the immune response to EBV.