RESUMO
Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes. We provided evidence that active proteasomes found within the lumen of phagosomes and endolysosomal vesicles locally generate antigenic peptides that can be directly loaded onto trafficking MHC-I molecules. However, the mechanism of active proteasome delivery to the endocytic compartments remained unknown. In this study, we demonstrate that phagosome-associated LC3A/B structures deliver proteasomes into subcellular compartments containing exogenous Ags and that autophagy drives TAP-independent, proteasome-dependent cross-presentation.
Assuntos
Apresentação Cruzada , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Apresentação de Antígeno , Autofagossomos , Fagossomos/metabolismo , Antígenos de Histocompatibilidade Classe I , Antígenos , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismoRESUMO
Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.
Assuntos
Anopheles , Animais , Anopheles/genética , Diferenciação Celular , Imunidade Inata/genética , Mosquitos Vetores/genética , Células GerminativasRESUMO
Gene-edited mosquitoes lacking a g amma-interferon-inducible lysosomal thiol reductase-like protein, namely ( mosGILT null ) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILT null A. gambiae was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILT null A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg , an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILT null mosquitoes. These results provide the crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.
RESUMO
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Evasão Tumoral , Humanos , Apresentação de Antígeno , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Neoplasias/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.
Assuntos
Núcleosídeo-Fosfato Quinase , Replicação Viral , Zika virus , Zika virus/fisiologia , Células Vero , Chlorocebus aethiops , Animais , Humanos , Núcleosídeo-Fosfato Quinase/metabolismo , Interferon Tipo I/metabolismo , Flavivirus/fisiologia , Mitocôndrias , Biossíntese de ProteínasRESUMO
Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.
Assuntos
COVID-19 , Antígenos de Histocompatibilidade Classe I , SARS-CoV-2 , Proteínas Virais Reguladoras e Acessórias , Humanos , Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Peptídeos , SARS-CoV-2/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin-MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the ß2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and ß2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I , Antígenos HLA-B , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulinas/metabolismo , Peptídeos/química , Ligação ProteicaRESUMO
Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).
Assuntos
Química Click , Proteômica , Animais , Linhagem Celular , Química Click/métodos , Mamíferos/metabolismo , Puromicina/análogos & derivados , Puromicina/farmacologiaRESUMO
Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein ß 2 -microglobulin (ß 2 m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 + T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8 + T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß 2 m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Significance Statement: Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with ß 2 m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of ß 2 m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.
RESUMO
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas , Antivirais/farmacologia , Imunidade Inata , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , S-Adenosilmetionina , Replicação ViralRESUMO
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
Assuntos
COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/genética , Linhagem Celular , Citocinas , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/química , Proteínas de Membrana/química , Modelos Moleculares , Proteínas de Neoplasias/química , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-AtividadeRESUMO
The endoplasmic reticulum (ER) performs key cellular functions including protein synthesis, lipid metabolism and signaling. While these functions are spatially isolated in structurally distinct regions of the ER, there is cross-talk between the pathways. One vital player that is involved in ER function is the ER-resident protein calreticulin (CALR). It is a calcium ion-dependent lectin chaperone that primarily assists in glycoprotein synthesis in the ER as part of the protein quality control machinery. CALR also buffers calcium ion release and mediates other glycan-independent protein interactions. Mutations in CALR have been reported in a subset of chronic blood tumors called myeloproliferative neoplasms. The mutations consist of insertions or deletions in the CALR gene that all cause a + 1 bp shift in the reading frame and lead to a dramatic alteration of the amino acid sequence of the C-terminal domain of CALR. This alters CALR function and affects cell homeostasis. This chapter will discuss how CALR and mutant CALR affect ER health and disease.
Assuntos
Retículo Endoplasmático , Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Genes Reguladores , Humanos , Mutação/genética , Transtornos Mieloproliferativos/genéticaRESUMO
The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.
Assuntos
COVID-19/imunologia , Exorribonucleases/imunologia , Evasão da Resposta Imune , Imunidade Inata , Biossíntese de Proteínas/imunologia , SARS-CoV-2/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Chlorocebus aethiops , Humanos , Células VeroRESUMO
Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.
Assuntos
Apresentação de Antígeno/genética , Infecções por HIV , Antígenos de Histocompatibilidade Classe I , Proteínas de Membrana Transportadoras , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Carga Viral/genética , Carga Viral/imunologiaRESUMO
Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine-co-ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency (R2 = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency in vitro also showed good transfection efficiency in vivo, and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.
Assuntos
Técnicas de Transferência de Genes , Luciferases de Renilla/genética , Sondas Moleculares/genética , RNA Mensageiro/genética , Citosol/química , Citosol/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Luciferases de Renilla/química , Sondas Moleculares/química , Nanopartículas/química , Transfecção/métodosRESUMO
Plasmodium infection in Anopheles is influenced by mosquito-derived factors. We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9). Here, we show that female mosaic mosGILT mutant mosquitoes display defects in ovarian development and refractoriness to Plasmodium. Following infection by either Plasmodium berghei or Plasmodium falciparum, mutant mosquitoes have significantly reduced oocyst numbers as a result of increased thioester-containing protein 1 (TEP1)-dependent parasite killing. Expression of vitellogenin (Vg), the major yolk protein that can reduce the parasite-killing efficiency of TEP1, is severely impaired in mutant mosquitoes. MosGILT is a mosquito factor that is essential for ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity.
Assuntos
Anopheles/genética , Anopheles/parasitologia , Malária/parasitologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Culicidae/genética , Feminino , Masculino , Camundongos , Mutação/genética , Plasmodium berghei/patogenicidade , Plasmodium falciparum/patogenicidade , Proteínas de Ligação a RNA/genética , Vitelogeninas/genéticaRESUMO
Viperin is an interferon (IFN)-inducible multifunctional protein. Recent evidence from high-throughput analyses indicates that most IFN-inducible proteins, including viperin, are intrinsically expressed in specific tissues; however, the respective intrinsic functions are unknown. Here we show that the intrinsic expression of viperin regulates adipose tissue thermogenesis, which is known to counter metabolic disease and contribute to the febrile response to pathogen invasion. Viperin knockout mice exhibit increased heat production, resulting in a reduction of fat mass, improvement of high-fat diet (HFD)-induced glucose tolerance, and enhancement of cold tolerance. These thermogenic phenotypes are attributed to an adipocyte-autonomous mechanism that regulates fatty acid ß-oxidation. Under an HFD, viperin expression is increased, and its function is enhanced. Our findings reveal the intrinsic function of viperin as a novel mechanism regulating thermogenesis in adipose tissues, suggesting that viperin represents a molecular target for thermoregulation in clinical contexts.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica , Proteínas/genética , Termogênese/genética , Adipócitos/metabolismo , Animais , Metabolismo Energético/genética , Masculino , Camundongos , Camundongos KnockoutRESUMO
During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.
Assuntos
Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/química , Complexo de Endopeptidases do Proteassoma/imunologia , Microglobulina beta-2/metabolismo , Animais , Apresentação de Antígeno , Células Cultivadas , Apresentação Cruzada , Células Dendríticas/citologia , Endocitose , Humanos , Camundongos , Fagossomos/imunologia , Proteólise , Microglobulina beta-2/genéticaRESUMO
My intention here is to describe the history of the molecular aspects of the antigen processing field from a personal perspective, beginning with the early identification of the species that we now know as MHC class I and MHC class II molecules, to the recognition that their stable surface expression and detection by T cells depends on peptide association, and to the unraveling of the biochemical and cell biological mechanisms that regulate peptide binding. One goal is to highlight the role that serendipity or, more colloquially, pure blind luck can play in advancing the research enterprise when it is combined with an appropriately receptive mind. This is not intended to be an overarching review, and because of my own work I focus primarily on studies of the human MHC. This means that I neglect the work of many other individuals who made advances in other species, particularly those who produced the many knockout mouse strains used to demonstrate the importance of the antigen processing machinery for initiating immune responses. I apologize in advance to colleagues around the globe whose contributions I deal with inadequately for these reasons, and to those whose foundational work is now firmly established in text books and therefore not cited. So many individuals have worked to advance the field that giving all of them the credit they deserve is almost impossible. I have attempted, while focusing on work from my own laboratory, to point out contemporaneous or sometimes earlier advances made by others. Much of the success of my own laboratory came because we simultaneously worked on both the MHC class I and class II systems and used the findings in one area to inform the other, but mainly it depended on the extraordinary group of students and fellows who have worked on these projects over the years. To those who worked in other areas who are not mentioned here, rest assured that I appreciate your efforts just as much.