RESUMEN
Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Biotecnología , ARN Guía de Sistemas CRISPR-Cas , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 has recently been identified as a critical tumour checkpoint, augmenting the expression and function of programmed death-ligand 1. We raised a monoclonal antibody, A9E8, specific for Siglec-15 using phage display. A9E8 stained myeloid leukaemia cell lines and peripheral cluster of differentiation (CD)33+ blasts and CD34+ leukaemia stem cells from patients with acute myeloid leukaemia (AML). By contrast, there was minimal expression on healthy donor leucocytes or CD34+ stem cells from non-AML donors, suggesting targeting Siglec-15 may have significant therapeutic advantages over its fellow Siglec CD33. After binding, A9E8 was rapidly internalised (half-life of 180 s) into K562 cells. Antibodies to Siglec-15 therefore hold therapeutic potential for AML treatment.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Inmunoglobulinas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Antígenos CD34/metabolismo , Femenino , Humanos , Células K562 , MasculinoRESUMEN
We recently discovered that TAPBPR promotes reglucosylation of the N-linked glycan on MHC class I molecules, a modification that restores their recognition by calreticulin and reincorporation into the peptide-loading complex. We wondered whether TAPBPR displayed some degree of glycan specificity, as is known to be the case for tapasin via its interaction with calreticulin & ERp57, or whether its interaction with MHC class I was glycan independent. Here, we explored this by comparing the ability of TAPBPR to bind to MHC class I containing either an intact or disrupted NxS/T glycosylation consensus sequence. In contrast to tapasin, TAPBPR bound strongly to MHC class I molecules that lacked N-linked glycosylation, suggesting that the TAPBPR:MHC class I interaction is glycan independent. Furthermore, we found that glycosylated HLA-A2 preferentially interacts with tapasin rather than TAPBPR, possibly explaining, in part, why MHC class I molecules bind efficiently to tapasin in the face of an alternative chaperone. The distinction in glycan specificity between the two peptide editors suggests that TAPBPR may bind to MHC class I molecules that are associated with a broader diversity of oligosaccharides attached compared with tapasin. This may explain, to some extent, the ability of TAPBPR to interact with MHC class I molecules outside of the ER.
Asunto(s)
Genes MHC Clase I/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Calreticulina/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Glicosilación , Células HeLa , Humanos , Proteínas de Transporte de Membrana/metabolismo , Péptidos/metabolismo , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Cápsulas Bacterianas/metabolismo , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Proteínas HSP90 de Choque Térmico/metabolismo , Tolerancia Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Animales , Línea Celular , Infecciones por Enterobacteriaceae/microbiología , Microbioma Gastrointestinal/inmunología , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , UbiquitinaciónRESUMEN
Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Proteínas de la Membrana/inmunología , Simulación del Acoplamiento Molecular , Péptidos/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Sitios de Unión/genética , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/metabolismo , Leucina/química , Leucina/inmunología , Leucina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Péptidos/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Proteínas de la Membrana/inmunología , Péptidos/inmunología , Animales , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunidad Celular , Inmunoglobulinas/genética , Interferón gamma/genética , Interferón gamma/inmunología , Células MCF-7 , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Péptidos/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
The presentation of antigenic peptides by MHC class I molecules plays a vital role in generating T cell responses against infection and cancer. Over the last two decades the central role of tapasin as a peptide editor that influences the loading and optimisation of peptides onto MHC class I molecules has been extensively characterised. Recently, it has become evident that the tapasin-related protein, TAPBPR, functions as a second peptide editor which influences the peptides displayed by MHC class I molecules. Here, we review the discovery of TAPBPR and current understanding of this novel protein in relation to its closest homologue tapasin.
Asunto(s)
Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Subgrupos de Linfocitos T/metabolismo , Animales , Presentación de Antígeno/inmunología , Microambiente Celular/inmunología , Epítopos/química , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptidos/química , Péptidos/inmunología , Polimorfismo Genético , Unión Proteica , Isoformas de Proteínas , Transducción de Señal , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex.
Asunto(s)
Presentación de Antígeno , Glucosiltransferasas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Línea Celular , Humanos , Mapeo de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Antígenos/metabolismo , Línea Celular , Humanos , Péptidos/metabolismo , Unión ProteicaRESUMEN
The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-ß expression. Influenza virus leads to induction of IFN-ß that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Regulación de la Expresión Génica/inmunología , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Mucosa Respiratoria/inmunología , Proteína 58 DEAD Box , Células Epiteliales/inmunología , Células Epiteliales/patología , Células Epiteliales/virología , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Gripe Humana/patología , Estructura Terciaria de Proteína , Receptores Inmunológicos , Mucosa Respiratoria/patología , Mucosa Respiratoria/virologíaRESUMEN
Ag presentation to CD4(+) and CD8(+) T cells depends on MHC class II and MHC class I molecules, respectively. One important regulatory factor of this process is the transcriptional regulation of MHC gene expression. It is well established that MHC class II transcription relies on the NLR protein CIITA. Recently, another NLR protein, NLRC5, was shown to drive MHC class I expression. The molecular mechanisms of the function of NLRC5 however remain largely elusive. In this study, we present a detailed functional study of the domains of NLRC5 revealing that the N-terminal domain of human NLRC5 has intrinsic transcriptional activity. Domain swapping experiments between NLRC5 and CIITA showed that this domain contributes to MHC class I and MHC class II gene expression with a bias for activation of MHC class I promoters. Delivery of this construct by adeno-associated viral vectors upregulated MHC class I and MHC class II expression in human cells and enhanced lysis of melanoma cells by CD8(+) cytotoxic T cells in vitro. Taken together, this work provides novel insight into the function of NLRC5 and CIITA in MHC gene regulation.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Linfocitos T Citotóxicos/inmunología , Activación Transcripcional/genética , Animales , Línea Celular Tumoral , Dependovirus/genética , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/genética , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Transactivadores/genéticaRESUMEN
Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I. In this review we detail the development of our understanding of NLRC5 function, discussing both the accepted and the controversial aspects of NLRC5 activity. We give insight into the molecular mechanisms, and the potential implications, of NLRC5 function in health and disease.
RESUMEN
NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Técnicas In Vitro , Microscopía Fluorescente , Proteína Adaptadora de Señalización NOD2/genética , Proteínas Nucleares/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.
Asunto(s)
Elementos de Facilitación Genéticos/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos HLA-A/fisiología , Antígenos HLA-B/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Transactivadores/fisiología , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Células HEK293 , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Transactivadores/antagonistas & inhibidores , Transactivadores/deficiencia , Activación Transcripcional/genéticaRESUMEN
Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.