RESUMEN
T cells express a somatically recombined antigen receptor (αßTCR) that is calibrated during development to respond to changes in peptides displayed by major histocompatibility complex proteins (pMHC) on the surface of antigen-presenting cells (APC). A key characteristic of pMHC for adaptive immunity is the ability to sample internal states of cells and tissues to sensitively detect changes associated with infection, cell derangement, or tissue injury. Physical T cell-APC contact sets up an axis for polarization of TCR, adhesion molecules, kinases, cytoskeletal elements, and organelles inherent in this mode of juxtacrine signaling. The discovery of further lateral organization of the TCR and adhesion molecules into radially symmetric compartments, the immunological synapse, revealed an intersecting plane of symmetry and potential for regulated symmetry breaking to control duration of T cell-APC interactions. In addition to organizing signaling machinery, the immunological synapse directs the polarized transport and secretion of cytokines and cytolytic agents across the synaptic cleft and is a site for the generation and exocytic release of bioactive microvesicles that can functionally affect recipient APC and other cells in the environment. This machinery is coopted by retroviruses, and human immune deficiency virus-1 may even use antigen-specific synapses for infection of healthy T cells. Here, we discuss recent advances in the molecular and cell biological mechanisms of immunological synapse assembly and signaling and its role in intercellular communication across the synaptic cleft.
Asunto(s)
Comunicación Celular , Sinapsis Inmunológicas/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Citoesqueleto/metabolismo , Infecciones por VIH/patología , HumanosRESUMEN
The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Polaridad Celular , Sinapsis Inmunológicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Vesículas Secretoras/metabolismo , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , VIH/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Sinapsis Inmunológicas/ultraestructura , Molécula 1 de Adhesión Intercelular/metabolismo , Activación de Linfocitos , Masculino , Ratones , Unión Proteica , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/ultraestructura , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Liberación del Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Agonist MHC-peptide complexes in the immunological synapse (IS) signal through T cell receptor (TCR) microclusters (MCs) that converge into a central supramolecular activation cluster (cSMAC). The determinants and function of the cSMAC remain unknown. We demonstrate an essential role for ubiquitin (Ub) and TSG101, but less so for HRS, in signal processing events at the cSMAC. Using siRNA in primary T cells, we show that Ub recognition by TSG101 is required for cSMAC formation, TCR MC signal termination, TCR downregulation, and segregation of TCR-MHC-peptide from PKC-theta-enriched signaling complexes. Weak agonist MHC-peptide induced CD80-dependent TCR MCs that dissociated in the center of the IS without recruiting TSG101. These results support TSG101-dependent recognition of CD80-independent TCR MCs as a molecular checkpoint for TCR downregulation.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Sinapsis Inmunológicas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Animales , Linfocitos B/inmunología , Antígeno B7-1/inmunología , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Humanos , Sinapsis Inmunológicas/metabolismo , Ratones , Fosforilación , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Factores de Transcripción/genéticaRESUMEN
T-cell receptor (TCR) triggering results in a cascade of intracellular tyrosine phosphorylation events that ultimately leads to T-cell activation. It is dependent on changes in the relative activities of membrane-associated tyrosine kinases and phosphatases near the engaged TCR. CD45 and CD148 are transmembrane tyrosine phosphatases with large ectodomains that have activatory and inhibitory effects on TCR triggering. This study investigates whether and how the ectodomains of CD45 and CD148 modulate their inhibitory effect on TCR signaling. Expression in T cells of forms of these phosphatases with truncated ectodomains inhibited TCR triggering. In contrast, when these phosphatases were expressed with large ectodomains, they had no inhibitory effect. Imaging studies revealed that truncation of the ectodomains enhanced colocalization of these phosphatases with ligated TCR at the immunological synapse. Our results suggest that the large ectodomains of CD45 and CD148 modulate their inhibitory effect by enabling their passive, size-based segregation from ligated TCR, supporting the kinetic-segregation model of TCR triggering.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Interleucina-2/metabolismo , Antígenos Comunes de Leucocito/química , Antígenos Comunes de Leucocito/genética , Activación de Linfocitos/genética , Ratones , Modelos Químicos , Mutagénesis/inmunología , Unión Proteica/inmunología , Estructura Terciaria de Proteína/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Relación Estructura-ActividadRESUMEN
Tumor antigen recognition by chimeric antigen receptors (CAR) triggers phosphorylation of their cytoplasmic portions resulting in CAR-T cell activation. We and others have shown that immunoreceptor triggering depends on the formation of close synaptic contacts, determined by the span of immunoreceptor-ligand complexes, from which large inhibitory phosphatases such as CD45 are sterically excluded. Here, we show, varying CAR-antigen complex span, that CAR-T cell activation depends on a formation of close contacts with target cells. CAR-antigen complexes with a span of 4 immunoglobulin superfamily (IgSF) domains maximize CAR-T cell activation, closely matching the span of endogenous TCR-pMHC complexes. Longer CAR-antigen complexes precipitously reduced triggering and cytokine production, but notably, anti-tumor cytotoxicity was largely preserved due to a â¼10-fold lower signaling threshold for mobilization of cytolytic effector function. Increased intermembrane spacing disrupted short-spanned PD-1-PD- L1 interactions, reducing CAR-T cell exhaustion. Together, our results show that membrane positioning across the immunological synapse can be engineered to generate CAR-T cells with clinically desirable functional profiles in vitro and in vivo .
RESUMEN
Gamma/delta (γδ) T cells are unconventional adaptive lymphocytes that recognize structurally diverse ligands via somatically-recombined antigen receptors (γδ TCRs). The molecular mechanism by which ligand recognition initiates γδ TCR signaling, a process known as TCR triggering, remains elusive. Unlike αß TCRs, γδ TCRs are not mechanosensitive, and do not require coreceptors or typical binding-induced conformational changes for triggering. Here, we show that γδ TCR triggering by nonclassical MHC class Ib antigens, a major class of ligands recognized by γδ T cells, requires steric segregation of the large cell-surface phosphatases CD45 and CD148 from engaged TCRs at synaptic close contact zones. Increasing access of these inhibitory phosphatases to sites of TCR engagement, by elongating MHC class Ib ligands or truncating CD45/148 ectodomains, abrogates TCR triggering and T cell activation. Our results identify a critical step in γδ TCR triggering and provide insight into the core triggering mechanism of endogenous and synthetic tyrosine-phosphorylated immunoreceptors.
RESUMEN
We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Antígenos H-2/inmunología , Nucleoproteínas/inmunología , Orthomyxoviridae/inmunología , Animales , Células CHO , Cricetinae , Cricetulus , Ratones , Ratones Endogámicos C57BL , Microglobulina beta-2/inmunologíaRESUMEN
The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR-pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial. It has been proposed that TCR triggering is promoted by segregation at the T cell-APC interface of cell-surface molecules with small ectodomains (such as TCR-pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation. A similar dependence on receptor-ligand complex dimensions was observed with artificial TCR-ligand systems that span the same dimensions as the TCR-pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR-pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering.
Asunto(s)
Antígenos de Histocompatibilidad/metabolismo , Activación de Linfocitos , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/química , Antígenos/inmunología , Células CHO , Bovinos , Línea Celular , Cricetinae , Antígenos de Histocompatibilidad/inmunología , Cinética , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Ligandos , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/inmunología , Animales , Complejo CD3/genética , Complejo CD3/inmunología , Complejo CD3/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Péptidos , Fosforilación/genética , Fosforilación/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/enzimología , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
T cells recognize small fragments of microorganisms (antigens) on the surface of other cells using T cell antigen receptors. The mechanism by which these receptors signal into T cells is controversial, but two recent studies provide important new clues.
Asunto(s)
Antígenos/inmunología , Inmunidad Celular/inmunología , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunología , Antígenos/metabolismo , Humanos , Complejo Mayor de Histocompatibilidad/inmunologíaRESUMEN
Background: Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses. Here we examine the importance of γc for myeloid dendritic cell (DC) function. Methods: We utilize a combination of in vitro DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. Results: We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular cis associations or cytoskeletal reorganization following MHCII ligation. Conclusions: These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.
RESUMEN
Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles.
RESUMEN
Autoimmune hepatitis is initiated by CD4 T cells that recognize self-antigen. The effector cells differentiate into functional phenotypes according to cytokines in the microenvironment and the nature of the triggering antigen. Hepatocytes can express class II molecules and present antigenic peptides through a bystander mechanism. NKT cells reside in the normal liver but may be involved in liver cell damage possibly through the expression of Fas ligand. Autoantibodies may also participate in the process by complexing with antigen on the hepatocyte membrane surface and by interacting with mononuclear cells with Fc receptors. Molecular mimicry generates cross-reactivities that predispose the immune system to cross-react with self-antigens. Multiple exposures to pathogens with antigenic similarities may prime a cross-reactive subset of T cells in the genetically predisposed host. Autoimmunity against one self-antigen may then spread through molecular mimicry to other homologous self-antigens and may lead to overt autoimmune disease. Anatomically distant tissues may also become involved as autoreactive T cells that had been activated in one organ expand and infiltrate other organs that express similar epitopes.
Asunto(s)
Hepatitis Autoinmune/etiología , Formación de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Antígenos HLA-DR/inmunología , Hepatitis Autoinmune/genética , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/patología , Humanos , Inmunidad Celular/inmunología , Modelos Inmunológicos , Imitación Molecular/inmunología , Autotolerancia/inmunologíaRESUMEN
Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Sinapsis Inmunológicas/inmunología , Esclerosis Múltiple/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular/inmunología , Antígenos HLA/inmunología , Humanos , Immunoblotting , Molécula 1 de Adhesión Intercelular/inmunología , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , FosforilaciónRESUMEN
Sub-micron scale signaling domains induced in the plasma membrane of cells are thought to play important roles in signal transduction. In T cells, agonist MHC-peptide complexes induce small diffraction-limited domains enriched in T cell receptor (TCR) and signaling molecules. These microclusters serve as transient platforms for signal initiation and are required for sustained signaling in T cells, although each microcluster functions for only a couple of minutes. How they are formed, and what mechanisms promote and regulate signaling within TCR microclusters is largely unknown, although it is clear that TCR engagement and dynamic reorganization of cortical actin are involved. Here, we review current understanding of signaling within microclusters in T cells, and speculate on how these structures may form, initiate biochemical signals, and serve as sites of both signal integration and amplification, while also facilitating appropriate termination of TCR and related signaling.
Asunto(s)
Transducción de Señal , Linfocitos T/metabolismo , Animales , HumanosRESUMEN
Despite intensive investigation we still do not understand how the T cell antigen receptor (TCR) tranduces signals across the plasma membrane, a process referred to as TCR triggering. Three basic mechanisms have been proposed, involving aggregation, conformational change, or segregation of the TCR upon binding pMHC ligand. Given the low density of pMHC ligand it remains doubtful that TCR aggregation initiates triggering, although it is likely to enhance subsequent signalling. Structural studies to date have not provided definitive evidence for or against a conformational change mechanism, but they have ruled out certain types of conformational change. Size-induced segregation of the bound TCR from inhibitory membrane tyrosine phosphatases seems to be required, but is probably not the only mechanism. Current evidence suggests that TCR triggering is initiated by a combination of segregation and conformational change, with subsequent aggregation contributing to amplification of the signal.
Asunto(s)
Membrana Celular/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Unión ProteicaRESUMEN
Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.
Asunto(s)
Uniones Adherentes/fisiología , Moléculas de Adhesión Celular/química , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Uniones Adherentes/química , Uniones Adherentes/ultraestructura , Secuencia de Aminoácidos , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/química , Membrana Celular/enzimología , Secuencia Conservada , Dimerización , Fibronectinas/química , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a ReceptoresRESUMEN
Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193-212, 238-257, 268-287, and 478-497). CYP2D6(193-212) is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6(193-212) arise through cross-reactive immunity between virus and self. We identified a hexameric sequence "RLLDLA" sharing 5 of 6 aa with "RLLDLS" of HCV(2985-2990) and all 6 aa with CMV(130-135). Of 17 CYP2D6(193-212)-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6(193-212) was inhibited by preincubation with HCV(2977-2996) or CMV(121-140). Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6(193-212). Affinity-purified CYP2D6(193-212)-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6(193-212) and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.