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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger-type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase-like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8(+) T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8(+) T cells but not that of naive CD4(+) T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.
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Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/inmunología , Activación de Linfocitos/inmunología , Lisina/inmunología , Proteína Tirosina Quinasa ZAP-70/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Activación de Linfocitos/genética , Lisina/genética , Lisina/metabolismo , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Fosforilación/inmunología , Poliubiquitina/inmunología , Poliubiquitina/metabolismo , Unión Proteica/inmunología , Interferencia de ARN , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Transcriptoma/genética , Transcriptoma/inmunología , Ubiquitina-Proteína Ligasas , Ubiquitinación/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Living organisms have evolved conserved 'catchers' for detecting and handling molecular patterns from invading viruses. Slavik et al. and Holleufer et al. recently identified cyclic GMP-AMP (cGAMP) synthase (cGAS)-like receptors that detect double-stranded viral RNA in Drosophila. cGAS-like receptors constitute a new expanding family of pattern recognition receptors (PRRs) for viral nucleic acids.
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Ácidos Nucleicos , Virus , Humanos , Nucleótidos Cíclicos , Nucleotidiltransferasas , Receptores de Reconocimiento de PatronesRESUMEN
BACKGROUND: Pathological retinal angiogenesis resulting from a variety of ocular diseases including oxygen induced retinopathy, diabetic retinopathy and ocular vein occlusion, is one of the major reasons for vision loss, yet the therapeutic option is limited. Multiple nanoparticles have been reported to alleviate angiogenic retinopathy. However, the adverse effect cannot be ignored due to the relatively large scale. Graphene quantum dots (GQDs) have shown potential in drug delivery and have been proved biocompatible. In this study, Graphene quantum dots are extensively investigated for their application in angiogenic retinopathy therapy. RESULTS: We showed that GQDs were biocompatible nanomaterials in vitro and in vivo. The nanoparticles have a dose-dependent inhibitory effect on proliferation, migration, tube formation and sprouting of human umbilical vein endothelial cells (HUVECs). Further data show that GQDs could inhibit pathological retinal neovascularization in an oxygen-induced retinopathy (OIR) model. The data of RNA sequencing suggested that periostin is involved in this process. GQDs inhibit the expression of periostin via STAT3, and further regulated cell cycle-related protein levels through ERK pathway. The signaling pathway was conformed in vivo using OIR mouse model. CONCLUSIONS: The present study indicated that GQDs could be a biocompatible anti-angiogenic nanomedicine in the treatment of pathological retinal neovascularization via disrupting periostin/ERK pathway and subsequent cell cycle.
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Grafito , Puntos Cuánticos , Enfermedades de la Retina , Animales , Proliferación Celular , Células Cultivadas , Grafito/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Puntos Cuánticos/uso terapéutico , Transducción de SeñalRESUMEN
E3 ubiquitin ligases are important in both innate and adaptive immunity. Here we report that Nrdp1, an E3 ubiquitin ligase, inhibited the production of proinflammatory cytokines but increased interferon-beta production in Toll-like receptor-triggered macrophages by suppressing adaptor MyD88-dependent activation of transcription factors NF-kappaB and AP-1 while promoting activation of the kinase TBK1 and transcription factor IRF3. Nrdp1 directly bound and polyubiquitinated MyD88 and TBK1, which led to degradation of MyD88 and activation of TBK1. Knockdown of Nrdp1 inhibited the degradation of MyD88 and the activation of TBK1 and IRF3. Nrdp1-transgenic mice showed resistance to lipopolysaccharide-induced endotoxin shock and to infection with vesicular stomatitis virus. Our data suggest that Nrdp1 functions as both an adaptor protein and an E3 unbiquitin ligase to regulate TLR responses in different ways.
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Proteínas Portadoras/metabolismo , Interferón Tipo I/metabolismo , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Células Cultivadas , Femenino , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/virología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/virología , Sepsis/inducido químicamente , Sepsis/metabolismo , Ubiquitina-Proteína Ligasas/genética , Vesiculovirus/fisiologíaRESUMEN
Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.
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Vacunas contra el Cáncer/inmunología , Carcinoma Pulmonar de Lewis/prevención & control , Quimiocinas/metabolismo , Exosomas/metabolismo , Trastornos de Estrés por Calor/prevención & control , Melanoma Experimental/prevención & control , Microdominios de Membrana/inmunología , Transducción de Señal/inmunología , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/metabolismo , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Células Cultivadas , Quimiocinas/administración & dosificación , Quimiocinas/inmunología , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Exosomas/inmunología , Trastornos de Estrés por Calor/inmunología , Trastornos de Estrés por Calor/metabolismo , Calor , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.
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Mediadores de Inflamación/fisiología , Interleucina-6/biosíntesis , Macrófagos Peritoneales/inmunología , Receptores Inmunológicos/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 9/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Ubiquitinación/inmunología , Animales , Línea Celular , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Uncontrolled viral replication and excessive inflammation are the main causes of death in the host infected with virus. Hence inhibition of intracellular viral replication and production of innate cytokines, which are the key strategies of hosts to fight virus infections, need to be finely tuned to eliminate viruses while avoid harmful inflammation. The E3 ligases in regulating virus replication and subsequent innate cytokines production remain to be fully characterized. Here we report that the deficiency of the E3 ubiquitin-protein ligase HECTD3 results in accelerated RNA virus clearance and reduced inflammatory response both in vitro and in vivo. Mechanistically, HECTD3 interacts with dsRNA-dependent protein kinase R (PKR) and mediates Lys33-linkage of PKR, which is the first non-proteolytic ubiquitin modification for PKR. This process disrupts the dimerization and phosphorylation of PKR and subsequent EIF2α activation, which results in the acceleration of virus replication, but promotes the formation of PKR-IKK complex and subsequent inflammatory response. The finding suggests HECTD3 is the potential therapeutic target for simultaneously restraining RNA virus replication and virus-induced inflammation once pharmacologically inhibited.
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Virus ARN , Virus , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Fosforilación , Replicación Viral , Citocinas/metabolismo , ARN Bicatenario/genética , Virus/genética , Virus/metabolismo , Proteínas Quinasas/metabolismo , Inflamación , Virus ARN/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
The endoplasmic reticulum (ER)-localized stimulator of interferon genes (STING) is the core adaptor for the pathogenic-DNA-triggered innate response. Aberrant activation of STING causes autoinflammatory and autoimmune diseases, raising the concern about how STING is finely tuned during innate response to pathogenic DNAs. Here, we report that the transmembrane domain (TM)-containing ER-localized E3 ubiquitin ligase TRIM13 (tripartite motif containing 13) is required for restraining inflammatory response to pathogenic DNAs. TRIM13 deficiency enhances pathogenic-DNA-triggered inflammatory cytokine production, inhibits DNA virus replication, and causes age-related autoinflammation. Mechanistically, TRIM13 interacts with STING via the TM and catalyzes Lys6-linked polyubiquitination of STING, leading to decelerated ER exit and accelerated ER-initiated degradation of STING. STING deficiency reverses the enhanced innate anti-DNA virus response in TRIM13 knockout mice. Our study delineates a potential strategy for controlling the homeostasis of STING by transmembrane ER-associated TRIM13 during the pathogenic-DNA-triggered inflammatory response.
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Retículo Endoplásmico , Ubiquitina-Proteína Ligasas , Animales , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Ratones , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
RNA-binding proteins (RBPs) play important roles in cancer development and treatment. However, the tumor-promoting RBPs and their partners, which may potentially serve as the cancer therapeutic targets, need to be further identified. Here, we report that zinc finger CCHC domain-containing protein 4 (ZCCHC4) is of aberrantly high expression in multiple human cancer tissues and is associated with poor prognosis and chemoresistance in patients of hepatocellular carcinoma (HCC), pancreatic cancer and colon cancer. ZCCHC4 promotes chemoresistance of HCC cells to DNA-damage agent (DDA) both in vitro and in vivo. HCC cell deficiency of ZCCHC4 reduces tumor growth in vivo and intratumoral interference of ZCCHC4 expression obviously enhances the DDA-induced antitumor effect. Mechanistically, ZCCHC4 inhibits DNA-damage-induced apoptosis in HCC cells by interacting with a new long noncoding RNA (lncRNA) AL133467.2 to hamper its pro-apoptotic function. Also, ZCCHC4 blocks the interaction between AL133467.2 and γH2AX upon DDA treatment to inhibit apoptotic signaling and promote chemoresistance to DDAs. Knockout of ZCCHC4 promotes AL133467.2 and γH2AX interaction for enhancing chemosensitivity in HCC cells. Together, our study identifies ZCCHC4 as a new predictor of cancer poor prognosis and a potential target for improving chemotherapy effects, providing mechanistic insights to the roles of RBPs and their partners in cancer progression and chemoresistance.
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Carcinoma Hepatocelular , Daño del ADN , Neoplasias Hepáticas , Metiltransferasas , ARN Largo no Codificante , Apoptosis/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , ADN/genética , ADN/metabolismo , Resistencia a Antineoplásicos , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
We identified human phosphatidylethanolamine-binding protein 4 (hPEBP4) as a human-derived novel member of the phosphatidylethanolamine-binding protein family, which is involved in apoptosis resistance of tumor cells. Because of its preferential expression in estrogen-related cancers, we wondered whether hPEBP4 plays a role in estrogen-induced cancer cell growth. Here, we demonstrated that hPEBP4 inhibited the 17beta-estradiol (E(2))-induced, proteasome-dependent estrogen receptor alpha (ERalpha) degradation to increase the protein level of ERalpha. Silencing of hPEBP4 inhibited the recruitment of ERalpha to the promoter of the ERalpha target gene pS2 in MCF-7 breast cancer cells after E(2) treatment. E(2)-induced, ERalpha-mediated transcription via the estrogen-response element, as well as the cellular proliferation, was significantly suppressed in hPEBP4-silenced MCF-7 cells. We found that Src, whose association with ERalpha facilitates the ERalpha binding to components of proteolytic machinery, could associate with hPEBP4 and that overexpression of hPEBP4 prevented the E(2)-induced interaction between ERalpha and Src. ERalpha overexpression, proteasome inhibitor, or Src inhibitor could reverse the suppression of ERalpha-mediated transactivation by hPEBP4 silencing. The inhibition of the proteasome degradation and the promotion of transactivation of ERalpha by hPEBP4 via the Src pathway were further confirmed in HeLa cells. Finally, we found that the promoting effects of hPEBP4 on ERalpha-mediated transactivation and estrogen-induced proliferation of cancer cells did not depend on its regulation of Akt and ERK activity. Our data suggest that hPEBP4 inhibits proteasome-dependent ERalpha degradation through the Src pathway, thus enhancing ERalpha-mediated transactivation and promoting the proliferation of cancer cells in response to estrogen.
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Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Activación Transcripcional/genética , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Estradiol/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
HSP are abundant and conserved proteins present in all cells. Upon temperature shock or other stress stimuli, HSP are synthesized intracellularly, which may protect cells from protein denaturation or from death. Although HSP are synthesized intracellularly, HSP can also be mobilized to the plasma membrane or even be released under stress conditions. Elucidating the roles of cell surface and extracellular HSP in immune regulation has attracted much attention in recent years. Extracellularly, HSP can serve a cytokine function to initiate both innate and adaptive immunity through activation of APC. HSP serves also a chaperone function and facilitates presentation of antigen peptide to T cells. Similarly, cell surface HSP may activate APC and promote antigen presentation through cell-cell contact. A study in this issue of the European Journal of Immunology demonstrates that cell surface HSP70 on DC induced by stress can upregulate membrane-associated IL-15, which in turn promotes the proliferation of CD4(+)CD45RA memory T cells. Moreover, a DC-CD4(+) T-cell interacting circuit formed by CD40L on T cells and CD40 on DC is proposed to play a role in the maintenance of memory homeostasis. This study has widened our view of HSP in adaptive immunity as well as their classical functions such as APC activator and antigen carrier.
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Inmunidad Adaptativa , Proteínas HSP70 de Choque Térmico/inmunología , Inmunidad Innata , Memoria Inmunológica , Animales , Linfocitos T CD4-Positivos , Células Dendríticas/inmunología , Humanos , Activación de Linfocitos/inmunologíaRESUMEN
Extracellular heat shock proteins (HSP) can activate dendritic cells (DC) and monocytes/macrophages, and HSP derived from tumor cells have been regarded as potent adjuvant facilitating presentation of tumor Ags and induction of antitumor immunity. However, the roles and the underlying mechanisms of releasable HSP in the induction of antitumor immunity have not been fully elucidated. In this study, we report that heat stress can induce the release of various HSP from tumor cells, which, in turn, activate tumor cells to produce chemokines for chemoattraction of DC and T cells via TLR4 signaling pathway. In vivo, we find that the infiltration and function of DC and T cells within tumor after local hyperthermia are increased significantly. We also provide evidence that HSP70 proteins released by tumor cells and TLR4 expressed by tumor cells/DC are essential for the chemoattraction of DC/T cells and for the subsequent induction of tumor-specific antitumor immunity. Therefore, our study suggests that heat stress-induced releasable HSP70 proteins from tumor cells play important roles in the initiation of antitumor immunity by inducing tumor cell production of chemokines and by activating the chemoattracted DC via TLR4 pathway.
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Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/prevención & control , Quimiocinas/biosíntesis , Células Dendríticas/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Calor , Transducción de Señal/inmunología , Receptor Toll-Like 4/fisiología , Animales , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Quimiocinas/fisiología , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Proteínas HSP70 de Choque Térmico/fisiología , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Fisiológico/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Cytokines produced by immune cells play pivotal roles in the regulation of both innate and adaptive immunity. However, the mechanisms controlling secretion of cytokines have not been fully elucidated. Secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane molecules implicated in regulating vesicular transport. In this study, we report the functional characterization of human SCAMP5 (hSCAMP5), a novel SCAMP protein that is widely expressed by a variety of neuronal and nonneuronal tissues and cells. By measuring the cytokine secretion (RANTES/CCL5 and IL-1beta) as an exocytotic model, we show that hSCAMP5 can promote the calcium-regulated signal peptide-containing cytokine (CCL5 but not IL-1beta) secretion in human epithelial cancer cells, human monocytes, and mouse macrophages. By using subcellular fractionation, immunofluorescence confocal microscopy, and membrane vesicle immunoisolation methods, we find that hSCAMP5 is mainly localized in the Golgi-associated compartments, and the calcium ionophore ionomycin can trigger a rapid translocation of hSCAMP5 from Golgi apparatus to plasma membrane along the classical exocytosis pathway. During the translocation of hSCAMP5 from Golgi apparatus to plasma membrane, hSCAMP5 can codistribute and complex with local soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) molecules. We further demonstrate that hSCAMP5 can directly interact with the calcium sensor synaptotagmins via the cytosolic C-terminal tail of hSCAMP5, thus providing a potential molecular mechanism linking SCAMPs with the SNARE molecules. Our findings suggest that hSCAMP5, in cooperation with the SNARE machinery, is involved in calcium-regulated exocytosis of signal peptide-containing cytokines.