RESUMO
In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.
Assuntos
Antígenos CD/imunologia , Linfócitos T/imunologia , Antígenos/imunologia , Antígenos CD/química , Antígenos CD/genética , Butirofilinas , Células Cultivadas , Difosfonatos/imunologia , Humanos , Imidazóis/imunologia , Espaço Intracelular , Ativação Linfocitária/genética , Mutação/genética , Ligação Proteica/genética , Engenharia de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Ácido ZoledrônicoRESUMO
Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.
Assuntos
Antígenos CD/química , Antígenos CD/imunologia , Butirofilinas/química , Butirofilinas/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Antígenos/química , Antígenos/imunologia , Antígenos CD/metabolismo , Butirofilinas/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes Quiméricas/imunologia , FosforilaçãoAssuntos
Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos , Linfócitos B/imunologia , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes , Citidina Desaminase/imunologia , Evolução Molecular Direcionada , Células HEK293 , Antígenos HLA/imunologia , Humanos , Hipermutação Somática de ImunoglobulinaRESUMO
Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.
Assuntos
Antígenos CD/metabolismo , Antígenos/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Anticorpos Bloqueadores , Anticorpos Imobilizados , Anticorpos Monoclonais , Antígenos CD/química , Antígenos CD/genética , Butirofilinas , Células Cultivadas , Células Clonais , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Fatores Imunológicos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/agonistas , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.
Assuntos
Bacteriófagos/genética , Proteínas do Capsídeo/metabolismo , Éxons , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Processamento Alternativo , Proteínas do Capsídeo/genética , Linhagem Celular , Clonagem Molecular , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Levivirus/genética , Proteínas de Ligação a Poli(A)/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Antígeno-1 Intracelular de Células TRESUMO
In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Rim/fisiologia , Proteínas de Ligação a Poli(A)/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ativação Transcricional/genética , Sítios de Ligação , Linhagem Celular , Humanos , Ligação Proteica , Sítios de Splice de RNA/genética , Antígeno-1 Intracelular de Células TRESUMO
Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vgamma9Vdelta2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vgamma9Vdelta2 T cell activation, we used stable beta(2)-microglobulin knockdown to generate tumor cells with a approximately 10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vgamma9Vdelta2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vgamma9Vdelta2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vgamma9Vdelta2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.
Assuntos
Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias/imunologia , Subpopulações de Linfócitos T/imunologia , Microglobulina beta-2/antagonistas & inibidores , Anticorpos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Regulação para Baixo , Humanos , Ativação Linfocitária , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores de Células Matadoras Naturais , Evasão Tumoral , Microglobulina beta-2/genéticaRESUMO
A considerable amount of smooth muscle phenotypic diversity is generated by tissue-specific and developmentally regulated splicing of alternative exons. The control mechanisms are unknown. We are using a myosin phosphatase targeting subunit-1 (MYPT1) alternative exon as a model to investigate this question. In the present study, we show that the RNA binding proteins TIA and PTB function as antagonistic enhancers and suppressors of splicing of the alternative exon, respectively. Each functions through a single U-rich element, containing two UCUU motifs, just downstream of the alternative exon 5' splice site. Tissue-specific down-regulation of TIA protein in the perinatal period allows PTB to bind to the U-rich element and suppress splicing of the alternative exon as the visceral smooth muscle acquires the fast-phasic smooth muscle contractile phenotype. This provides a novel role for PTB in the tissue-specific regulation of splicing of alternative exons during the generation of smooth muscle phenotypic diversity.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Éxons/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosfoproteínas Fosfatases/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Galinhas , Moela das Aves/metabolismo , Humanos , Contração Muscular , Músculo Liso/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ligação a Poli(A) , Proteína Fosfatase 1 , Ratos , Antígeno-1 Intracelular de Células TRESUMO
Alternative CD44 exons v8, v9, and v10 are spliced as a block in epithelial cells (for example SVK14 cells), but can be skipped as a block by other cells. Using a minigene approach, we show that downstream intronic UGG repeats participate in activation of v8 exon splicing in SVK14 cells. The repeats can activate splicing of a heterologous exon in SVK14 cells and act additively with a previously described v8 exon splicing enhancer in this context. An alternative v9 exon 5' splice site used by some cells to make an aberrant transcript is repressed by an immediately downstream (UGG)3 sequence in SVK14 cells. We conclude that UGG repeats both activate v8 exon splicing and repress use of the alternative v9 exon 5' splice site in SVK14 cells, thus participating in the coordination of correct epithelial cell splicing of the v8-10 block.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Receptores de Hialuronatos/genética , Íntrons/genética , Análise de Sequência de DNA/métodos , Repetições de Trinucleotídeos/genética , Sequência de Bases , DNA Recombinante/genética , Dados de Sequência MolecularRESUMO
TIA-1 and TIAR are a pair of related RNA-binding proteins which have been implicated in apoptosis. We show that chicken DT40 cells with both tia-1 alleles and one tiar allele disrupted (tia-1(-/-)tiar(-/+) cells) are viable. However, their growth and survival in medium containing low serum levels is significantly reduced compared with DT40 cells. The remaining intact tiar allele in tia-1(-/-)tiar(-/+) cells can only be disrupted if TIA-1 expression is first restored to the cells by transfection of a TIA-1 expression vector. We conclude that DT40 cells require either TIA-1 or TIAR for viability. TIA-1 overexpression in tia-1(-/-)tiar(-/+) cells leads to a radical drop in TIAR levels, by inducing efficient splicing of two tiar alternative exons carrying in-frame stop codons. In wild-type DT40 cells, tiar transcripts including these exons can also be detected. These transcripts increase significantly in abundance in cycloheximide-treated cells, suggesting that splicing of the exons exposes mRNAs to nonsense-mediated mRNA decay. TIA-1 or TIAR depletion leads to a marked drop in splicing of the exons. The human tiar gene contains a corresponding pair of TIA-1-inducible alternative exons, and we show that there is very high sequence conservation between chickens and humans of the exon pair and parts of the flanking introns. The TIA-1/TIAR responsiveness of these alternative tiar exons is likely to be of physiological importance for controlling TIAR levels.
Assuntos
Proteínas de Membrana/fisiologia , Proteínas , Proteínas de Ligação a RNA/fisiologia , Alelos , Animais , Apoptose , Sequência de Bases , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Galinhas , Códon , Éxons , Humanos , Íntrons , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Ligação a Poli(A) , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Antígeno-1 Intracelular de Células TRESUMO
We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.
Assuntos
Músculo Liso/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/genética , Proteínas/metabolismo , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Galinhas , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Moela das Aves/citologia , Moela das Aves/fisiologia , Dados de Sequência Molecular , Músculo Liso/citologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteínas/genética , Sítios de Splice de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Retículo Sarcoplasmático/metabolismoRESUMO
The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.