RESUMEN
Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnß transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnß transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Inmunidad Innata , Macrófagos/inmunología , Sirtuinas/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , ARN Helicasas DEAD-box/genética , Células HEK293 , Humanos , Lipoilación/genética , Lipoilación/inmunología , Macrófagos/virología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Células RAW 264.7 , Sirtuinas/genética , Estomatitis Vesicular/genética , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
Primary biliary cholangitis (PBC) is characterized by the presence of serum anti-mitochondrial autoantibodies (AMAs). To date, four antigens among the 2-oxo-acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA-corresponding antigens (AMA-antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA-antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano-liquid chromatography-tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein-search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA-antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well-known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Autoantígenos/inmunología , Lipoilación/inmunología , Cirrosis Hepática Biliar/inmunología , Mitocondrias/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/inmunología , Epítopos/inmunología , Femenino , Humanos , Persona de Mediana Edad , Complejo Piruvato Deshidrogenasa/inmunología , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/química , Lisofosfolípidos/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Antígenos de Histocompatibilidad Clase I/inmunología , Lipoilación/inmunología , Lisofosfolípidos/inmunología , Macaca mulatta , Péptidos/química , Péptidos/inmunología , Estructura Cuaternaria de Proteína , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
BACKGROUND AND AIMS: Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). METHODS: Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. RESULTS: The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. CONCLUSIONS: Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. LAY SUMMARY: Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.
Asunto(s)
Antígenos CD36/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Metabolismo de los Lípidos/inmunología , Lipoilación/inmunología , Hígado , Enfermedad del Hígado Graso no Alcohólico , Adenosina Monofosfato/metabolismo , Animales , Células Hep G2 , Humanos , Inflamación/metabolismo , Hígado/metabolismo , Hígado/patología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.
Asunto(s)
Exocitosis/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Vesículas Secretoras/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Citoesqueleto de Actina/inmunología , Exocitosis/efectos de los fármacos , Gelatinasas/inmunología , Humanos , Lipoilación/efectos de los fármacos , Lipoilación/inmunología , Proteína Quinasa C/inmunología , Proteínas Proto-Oncogénicas c-abl/inmunología , Proteínas Proto-Oncogénicas c-raf/inmunología , Factor de Necrosis Tumoral alfa/farmacología , alfa-Defensinas/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.
Asunto(s)
Apoptosis/inmunología , Autoinmunidad/inmunología , Microdominios de Membrana/inmunología , Ratones Transgénicos , Receptor fas/inmunología , Animales , Apoptosis/genética , Autoinmunidad/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Células HEK293 , Humanos , Lipoilación/inmunología , Microdominios de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Human germinal center-associated lymphoma (HGAL) is specifically expressed only in germinal center (GC) B lymphocytes and GC-derived lymphomas. HGAL protein decreases lymphocyte motility by inhibiting the ability of myosin to translocate actin via direct interaction with F-actin and myosin II and by activating RhoA signaling via direct interactions with RhoA-specific guanine nucleotide exchange factors. HGAL protein also regulates B-cell receptor (BCR) signaling by directly binding to and enhancing Syk kinase activity and activation of its downstream effectors. Herein we demonstrate that HGAL protein can be myristoylated and palmitoylated and that these modifications localize HGAL to cellular membrane raft microdomains with distinct consequences for BCR signaling and chemoattractant-induced cell mobility. In BCR signaling, raft localization of HGAL facilitates interaction with Syk and modulation of the BCR activation and signaling, which induces HGAL phosphorylation and redistribution from lipid raft to bulk membrane and cytoplasm, followed by degradation. In contrast, HGAL myristoylation and palmitoylation avert its inhibitory effects on chemoattractant-induced cell motility. These findings further elucidate the growing and complex role of HGAL in B-cell biology and suggest that membrane-bound and cytoplasmic HGAL protein differently regulates distinct biological processes.
Asunto(s)
Linfocitos B/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/fisiología , Actinas/genética , Actinas/inmunología , Actinas/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Factores Quimiotácticos/genética , Factores Quimiotácticos/inmunología , Factores Quimiotácticos/metabolismo , Citoplasma/genética , Citoplasma/inmunología , Citoplasma/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoilación/genética , Lipoilación/inmunología , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , Proteínas de Microfilamentos , Miosina Tipo II/genética , Miosina Tipo II/inmunología , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Transporte de Proteínas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Quinasa Syk , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/inmunología , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Although protein S-palmitoylation was first characterized >30 years ago, and is implicated in the function, trafficking, and localization of many proteins, little is known about the regulation and physiological implications of this posttranslational modification. Palmitoylation of various signaling proteins required for TCR-induced T cell activation is also necessary for their proper function. Linker for activation of T cells (LAT) is an essential scaffolding protein involved in T cell development and activation, and we found that its palmitoylation is selectively impaired in anergic T cells. The recent discovery of the DHHC family of palmitoyl acyl transferases and the establishment of sensitive and quantitative proteomics-based methods for global analysis of the palmitoyl proteome led to significant progress in studying the biology and underlying mechanisms of cellular protein palmitoylation. We are using these approaches to explore the palmitoyl proteome in T lymphocytes and, specifically, the mechanistic basis for the impaired palmitoylation of LAT in anergic T cells. This chapter reviews the history of protein palmitoylation and its role in T cell activation, the DHHC family and new methodologies for global analysis of the palmitoyl proteome, and summarizes our recent work in this area. The new methodologies will accelerate the pace of research and provide a greatly improved mechanistic and molecular understanding of the complex process of protein palmitoylation and its regulation, and the substrate specificity of the novel DHHC family. Reversible protein palmitoylation will likely prove to be an important posttranslational mechanism that regulates cellular responses, similar to protein phosphorylation and ubiquitination.
Asunto(s)
Aciltransferasas/inmunología , Lipoilación/inmunología , Procesamiento Proteico-Postraduccional , Linfocitos T/inmunología , Tioléster Hidrolasas/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Anergia Clonal , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Humanos , Lipoilación/genética , Activación de Linfocitos , Proteínas de la Membrana/inmunología , Proteómica , Tioléster Hidrolasas/genéticaRESUMEN
Ras transmits manifold signals from the TCR at various crossroads in the life of a T cell. For example, selection programs in the thymus or the acquisition of a state of hypo-responsiveness known as anergy are just some of the T cell features known to be controlled by TCR-sparked signals that are intracellularly propagated by Ras. These findings raise the question of how Ras can transmit such a variety of signals leading to the shaping of equally many T cell traits. Because Ras proteins transit through endomembrane compartments on their way to the plasma membrane (PM), compartmentalized Ras activation at distinct subcellular sites represents a potential mechanism for signal diversification in TCR signaling. This hypothesis has been nurtured by studies in T cells engineered to overexpress Ras that reported distinct activation of Ras at the PM and Golgi. Contrary to this scenario, we report in this study that activation of endogenous Ras, imaged in live Jurkat T cells using novel affinity probes for Ras-GTP, proceeds only at the PM even upon enforced signal flux through the diacylglycerol/RasGRP1 pathway. Physiological engagement of the TCR at the immunological synapse in primary T cells caused focalized Ras-GTP accumulation also only at the PM. Analysis of palmitoylation-deficient Ras mutants, which are confined to endomembranes, confirmed that the TCR does not activate Ras in that compartment and revealed a critical function for palmitoylation in N-Ras/H-Ras activation. These findings identify the PM as the only site of TCR-driven Ras activation and document that endomembranes are not a signaling platform for Ras in T cells.
Asunto(s)
Membrana Celular/metabolismo , Lipoilación/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Colorantes Fluorescentes/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Células Jurkat , Lipoilación/genética , Proteína Oncogénica p21(ras)/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Ratas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/biosíntesis , Factores de Intercambio de Guanina Nucleótido ras/genética , Proteínas ras/biosíntesisRESUMEN
Previous studies on the MHC class-specific differentiation of CD4(+)CD8(+) thymocytes into CD4(+) and CD8(+) T cells have focused on the role of coreceptor molecules. However, CD4 and CD8 T cells develop according to their MHC class specificities even in these mice lacking coreceptors. This study investigated the possibility that lineage is determined not only by coreceptors, but is also guided by the way how MHC molecules are presented. MHC class II molecules possess a highly conserved Cys in their transmembrane domain, which is palmitoylated and thereby associates with lipid rafts, whereas neither palmitoylation nor raft association was observed with MHC class I molecules. The generation of CD4 T cells was impaired and that of CD8 T cells was augmented when the rafts on the thymic epithelial cells were disrupted. This was due to the conversion of MHC class II-specific thymocytes from the CD4 lineage to CD8. The ability of I-A(d) molecule to associate with rafts was lost when its transmembrane Cys was replaced. The development of DO11.10 thymocytes recognizing this mutant I-A(dm) was converted from CD4 to CD8. These results suggest that the CD4 lineage commitment is directed by the raft-associated presentation of MHC class II molecules.