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1.
Immunity ; 54(1): 116-131.e10, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33271120

RESUMO

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.


Assuntos
Linfócitos B/fisiologia , Biomarcadores Tumorais/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Linfoma Difuso de Grandes Células B/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Estudo de Associação Genômica Ampla , Antígenos HLA/metabolismo , Humanos , Vigilância Imunológica , Linfoma Difuso de Grandes Células B/metabolismo , Evasão Tumoral/genética
2.
Mol Cell ; 73(6): 1162-1173.e5, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30712990

RESUMO

The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.


Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/biossíntese , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno , Humanos , Vigilância Imunológica , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Melanoma/imunologia , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/virologia
3.
Mol Cell ; 53(4): 562-576, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24508390

RESUMO

The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Regulação Enzimológica da Expressão Gênica , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Proliferação de Células , Cisteína/química , Dissulfetos/química , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Inositol/química , Camundongos , Dados de Sequência Molecular , Oxigênio/química , Fosforilação , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais
4.
J Immunol ; 198(10): 3835-3845, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28363906

RESUMO

CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I-restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I-based immunosurveillance.


Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Nucleosídeos/farmacologia , Peptídeos/imunologia , Sulfonamidas/farmacologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Citosol/química , Citosol/imunologia , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/imunologia , Cinética , Ligantes , Camundongos , Monitorização Imunológica , Peptídeos/metabolismo , Pirazóis , Pirimidinas , Sulfetos , Ubiquitinação , Vaccinia virus/química , Vaccinia virus/imunologia
5.
Proc Natl Acad Sci U S A ; 109(26): 10587-92, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22645345

RESUMO

Many age-related diseases are known to elicit protein misfolding and aggregation. Whereas environmental stressors, such as temperature, oxidative stress, and osmotic stress, can also damage proteins, it is not known whether aging and the environment impact protein folding in the same or different ways. Using polyQ reporters of protein folding in both Caenorhabditis elegans and mammalian cell culture, we show that osmotic stress, but not other proteotoxic stressors, induces rapid (minutes) cytoplasmic polyQ aggregation. Osmotic stress-induced polyQ aggregates could be distinguished from aging-induced polyQ aggregates based on morphological, biophysical, cell biological, and biochemical criteria, suggesting that they are a unique misfolded-protein species. The insulin-like growth factor signaling mutant daf-2, which inhibits age-induced polyQ aggregation and protects C. elegans from stress, did not prevent the formation of stress-induced polyQ aggregates. However, osmotic stress resistance mutants, which genetically activate the osmotic stress response, strongly inhibited the formation of osmotic polyQ aggregates. Our findings show that in vivo, the same protein can adopt distinct aggregation states depending on the initiating stressor and that stress and aging impact the proteome in related but distinct ways.


Assuntos
Envelhecimento/metabolismo , Peptídeos/metabolismo , Estresse Fisiológico , Animais , Camundongos , Pressão Osmótica , Estresse Oxidativo
6.
J Cell Sci ; 125(Pt 20): 4865-75, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22854046

RESUMO

ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.


Assuntos
Proteínas de Choque Térmico , Glicoproteínas de Membrana , Dobramento de Proteína , Resposta a Proteínas não Dobradas/genética , Animais , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Inativação Gênica , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Transdução de Sinais
7.
Elife ; 122024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38512721

RESUMO

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.


Assuntos
Biossíntese de Proteínas , Ribossomos , Camundongos , Animais , Ribossomos/metabolismo , Linfócitos , Citometria de Fluxo , Mamíferos
8.
Front Immunol ; 14: 1116906, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36761745

RESUMO

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain 'framework' residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities.


Assuntos
Antígenos de Histocompatibilidade Classe I , Receptores de Antígenos de Linfócitos T , Humanos , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Antígenos HLA , Regiões Determinantes de Complementaridade/química , Antígenos
9.
Semin Cell Dev Biol ; 21(5): 479-85, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20223290

RESUMO

A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).


Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Animais , Proteínas de Choque Térmico HSP70 , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
10.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393509

RESUMO

The success of tumor immunotherapy, while partial, confirms the existence and importance of tumor immunosurveillance. CD8+ T cell recognition of tumor-specific peptides bound to MHC class I (MHC-I) molecules is central to this process. In this issue of the JCI, Fang, Wang, et al. describe a unique tumor immunoevasion strategy based on endocytosis and degradation of MHC-I complexes mediated by the trafficking factor MAL2. Notably, MAL2 expression was associated with poor prognosis of breast cancer, and its downregulation enhanced CD8+ T cell recognition of breast cancer in various experimental models. This work demonstrates that a deeper understanding of tumor interference with MHC-I stability and trafficking has considerable potential for enhancing immunotherapies.


Assuntos
Neoplasias da Mama , Antígenos de Histocompatibilidade Classe I , Antígenos de Neoplasias , Linfócitos T CD8-Positivos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoterapia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina
11.
Nat Rev Immunol ; 21(2): 116-128, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32820267

RESUMO

The remarkable success of immune checkpoint inhibitors demonstrates the potential of tumour-specific CD8+ T cells to prevent and treat cancer. Although the number of lives saved by immunotherapy mounts, only a relatively small fraction of patients are cured. Here, we review two of the factors that limit the application of CD8+ T cell immunotherapies: difficulties in identifying tumour-specific peptides presented by MHC class I molecules and the ability of tumour cells to impair antigen presentation as they evolve under T cell selection. We describe recent advances in understanding how peptides are generated from non-canonical translation of defective ribosomal products, relate this to the dysregulated translation that is a feature of carcinogenesis and propose dysregulated translation as an important new source of tumour-specific peptides. We discuss how the synthesis and function of components of the antigen-processing and presentation pathway, including the recently described immunoribosome, are manipulated by tumours for immunoevasion and point to common druggable targets that may enhance immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune , Vigilância Imunológica , Neoplasias/imunologia , Apresentação de Antígeno , Humanos , Imunoterapia Adotiva , Neoplasias/terapia
12.
Genome Biol ; 22(1): 330, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34872593

RESUMO

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines.


Assuntos
Interferons , Nanoporos , Pseudouridina/genética , Pseudouridina/metabolismo , RNA Mensageiro/genética , Animais , Bactérias , Caenorhabditis elegans , Drosophila , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Aprendizado de Máquina , RNA , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNA/métodos , Transcriptoma
13.
Nat Rev Immunol ; 20(10): 644, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32873889

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

14.
Nat Struct Mol Biol ; 27(9): 814-821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719458

RESUMO

Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code embedded in 5' UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5' UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5' UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5' UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.


Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/genética , Células HEK293 , Humanos , Fases de Leitura Aberta , RNA Mensageiro/química
16.
Methods Mol Biol ; 1988: 109-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147936

RESUMO

Antigen presentation by classical MHC class I molecules to CD8+ T cells is a central aspect of the adaptive immune response. Here, we describe methods to monitor antigen presentation using the model ovalbumin Kb-binding peptide, SIINFEKL. SIINFEKL genetically incorporated into viral or cellular source proteins can be used to precisely probe various aspects of antigen presentation, including the kinetics of peptide generation, MHC class I surface stability, and presentation efficiency following pharmacological and genetic manipulations including genome wide and high throughput drug screening.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Biologia Molecular/métodos , Peptídeos/metabolismo , Linhagem Celular , Fluorescência , Humanos , Cinética , Plasmídeos/metabolismo
17.
Trends Cell Biol ; 29(12): 929-939, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31662235

RESUMO

MHC class I presentation of short peptides enables CD8+ T cell (TCD8+) immunosurveillance of tumors and intracellular pathogens. A key feature of the class I pathway is that the immunopeptidome is highly skewed from the cellular degradome, indicating high selectivity of the access of protease-generated peptides to class I molecules. Similarly, in professional antigen-presenting cells, peptides from minute amounts of proteins introduced into the cytosol outcompete an overwhelming supply of constitutively generated peptides. Here, we propose that antigen processing is based on substrate channeling and review recent studies from the antigen processing and cell biology fields that provide a starting point for testing this hypothesis.


Assuntos
Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vigilância Imunológica/imunologia , Imunidade Adaptativa/imunologia , Animais , Humanos , Peptídeos/imunologia
18.
PLoS One ; 13(7): e0200913, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30024926

RESUMO

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling.


Assuntos
Citocinas/metabolismo , Mutação , Receptor Toll-Like 9/química , Receptor Toll-Like 9/metabolismo , Tirosina/química , Animais , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Receptor Toll-Like 9/genética , Tirosina/genética
19.
Front Immunol ; 9: 2934, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619294

RESUMO

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14++/CD16+ intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response.


Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Interleucina/metabolismo , Resposta a Proteínas não Dobradas , Células Cultivadas , Células Dendríticas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Interleucinas/química , Interleucinas/genética , Interleucinas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Receptores de Interleucina/química , Receptores de Interleucina/genética , Transdução de Sinais/genética , Interleucina 22
20.
J Clin Invest ; 126(12): 4399-4401, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27841762

RESUMO

CD8+ T cells play a central role in eradicating intracellular pathogens, but also are important for noninfectious diseases, including cancer and autoimmunity. The ability to clinically manipulate CD8+ T cells to target cancer and autoimmune disease is limited by our ignorance of relevant self-peptide target antigens. In this issue of the JCI, Pearson et al. describe 25,270 MHC class I-associated peptides presented by a wide range of HLA A and B allomorphs expressed by 18 different B cell lines. Via extensive bioinformatic analysis, the authors make surprising conclusions regarding the selective nature of peptide generation at the level of individual gene products and create a predictive algorithm for disease-relevant self-peptides that will be of immediate use for clinical and basic immunological research.


Assuntos
Algoritmos , Doenças Autoimunes/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Peptídeos/imunologia , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Humanos , Neoplasias/patologia
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