RESUMO
Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodominant antigens is orchestrated by molecular chaperones: tapasin/TAP-binding protein, related for MHC-I and HLA-DM for MHC-II. Once peptide/MHC (pMHC) complexes are assembled, following various steps of quality control, they are delivered to the cell surface, where they are available for identification by αß receptors on CD8+ or CD4+ T lymphocytes. In addition, recognition of cell surface peptide/MHC-I complexes by natural killer cell receptors plays a regulatory role in some aspects of the innate immune response. Many of the components of the pathways of antigen processing and presentation and of T cell receptor (TCR)-mediated signaling have been studied extensively by biochemical, genetic, immunological, and structural approaches over the past several decades. Until recently, however, dynamic aspects of the interactions of peptide with MHC, MHC with molecular chaperones, or of pMHC with TCR have been difficult to address experimentally, although computational approaches such as molecular dynamics (MD) simulations have been illuminating. Studies exploiting X-ray crystallography, cryo-electron microscopy, and multidimensional nuclear magnetic resonance (NMR) spectroscopy are beginning to reveal the importance of molecular flexibility as it pertains to peptide loading onto MHC molecules, the interactions between pMHC and TCR, and subsequent TCR-mediated signals. In addition, recent structural and dynamic insights into how molecular chaperones define peptide selection and fine-tune the MHC displayed antigen repertoire are discussed. Here, we offer a review of current knowledge that highlights experimental data obtained by X-ray crystallography and multidimensional NMR methodologies. Collectively, these findings strongly support a multifaceted role for protein plasticity and conformational dynamics throughout the antigen processing and presentation pathway in dictating antigen selection and recognition.
RESUMO
As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the ß2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and ß2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Muromegalovirus/química , Proteínas do Envelope Viral/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Muromegalovirus/genética , Muromegalovirus/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the µ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining µ subunits in the cells are eventually able to reroute class I molecules to lysosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Complexo 1 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 3 de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Muromegalovirus/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
UNLABELLED: The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment. IMPORTANCE: In this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Infecções por Roseolovirus/metabolismo , Infecções por Roseolovirus/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas de Transporte/genética , Herpesvirus Humano 7/química , Herpesvirus Humano 7/genética , Humanos , Ligação Proteica , Multimerização Proteica , Proteínas Virais/genéticaRESUMO
Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells.
Assuntos
Antígenos CD1/biossíntese , Galactosilceramidas/imunologia , Glicoproteínas/biossíntese , Células T Matadoras Naturais/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD1/imunologia , Antígenos CD1/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Células HeLa , Humanos , Ativação Linfocitária/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules.
Assuntos
Proteínas de Transporte/metabolismo , Glioblastoma/metabolismo , Antígeno HLA-A2/metabolismo , Herpesvirus Humano 7/metabolismo , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Diferenciação Celular , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Imunofluorescência , Glicosilação , Antígeno HLA-A2/genética , Humanos , Imunoprecipitação , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Microglobulina beta-2/antagonistas & inibidores , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
Herpesviruses have evolved numerous strategies to evade detection by the immune system. Notably, most of the herpesviruses interfere with viral antigen presentation to cytotoxic T lymphocytes (CTLs) by removing class I major histocompatibility complex (MHC) molecules from the infected cell surface. Clearly, since the herpesviruses have evolved an extensive array of mechanisms to remove class I MHC molecules from the cell surface, this strategy serves them well. However, class I MHC molecules often serve as inhibitory ligands for NK cells, so viral downregulation of all class I MHC molecules should leave the infected cell open to NK cell attack. Some viruses solve this problem by selectively downregulating certain class I MHC products, leaving other class I products at the cell surface to serve as inhibitory NK cell ligands. Here, we show that human herpesvirus 7 (HHV-7) U21 binds to and downregulates all of the human class I MHC gene products, as well as the murine class I molecule H-2K(b). HHV-7-infected cells must therefore possess other means of escaping NK cell detection.
Assuntos
Proteínas de Transporte/fisiologia , Regulação para Baixo , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 7/patogenicidade , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas Virais/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5' or 3' showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3' to 5' or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.