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1.
J Cell Physiol ; 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31944299

RESUMO

Hypoxia occurs in physiological and pathological conditions. T cells experience hypoxia in pathological and physiological conditions as well as in lymphoid organs. Indeed, hypoxia-inducible factor 1α (HIF-1α) affects T cell survival and functions. Rai, an Shc family protein member, exerts pro-survival effects in hypoxic neuroblastoma cells. Since Rai is also expressed in T cells, we here investigated its role in hypoxic T cells. In this work, hypoxia differently affected cell survival, proapoptotic, and metabolic programs in T cells, depending upon Rai expression. By using Jurkat cells stably expressing Rai and splenocytes from Rai-/- mice, we demonstrated that Rai promotes T cell survival and affects cell metabolism under hypoxia. Upon exposure to hypoxia, Jurkat T cells expressing Rai show (a) higher HIF-1α protein levels; (b) a decreased cell death and increased Akt/extracellular-signal-regulated kinase phosphorylation; (c) a decreased expression of proapoptotic markers, including caspase activities and poly(ADP-ribose) polymerase cleavage; (d) an increased glucose and lactate metabolism; (e) an increased activation of nuclear factor-kB pathway. The opposite effects were observed in hypoxic splenocytes from Rai-/- mice. Thus, Rai plays an important role in hypoxic signaling and may be relevant in the protection of T cells against hypoxia.

2.
Cell Death Differ ; 27(1): 310-328, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31142807

RESUMO

The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.

3.
Front Cell Dev Biol ; 7: 292, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803744

RESUMO

Ciliated cells exploit a specific transport system, the intraflagellar transport (IFT) system, to ensure the traffic of molecules from the cell body to the cilium. However, it is now clear that IFT activity is not restricted to cilia-related functions. This is strikingly exemplified by the observation that IFT proteins play important roles in cells lacking a primary cilium, such as lymphocytes. Indeed, in T cells the IFT system regulates the polarized transport of endosome-associated T cell antigen receptors and signaling mediators during assembly of the immune synapse, a specialized interface that forms on encounter with a cognate antigen presenting cell and on which T cell activation and effector function crucially depend. Cellular degradation pathways have recently emerged as new extraciliary functions of the IFT system. IFT proteins have been demonstrated to regulate autophagy in ciliated cells through their ability to recruit the autophagy machinery to the base of the cilium. We have now implicated the IFT component IFT20 in another central degradation process that also controls the latest steps in autophagy, namely lysosome function, by regulating the cation-independent mannose-6-phosphate receptor (CI-MPR)-dependent lysosomal targeting of acid hydrolases. This involves the ability of IFT20 to act as an adaptor coupling the CI-MPR to dynein for retrograde transport to the trans-Golgi network. In this short review we will discuss the emerging roles of IFT proteins in cellular degradation pathways.

4.
Eur J Immunol ; 49(10): 1457-1973, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31633216

RESUMO

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

5.
Cells ; 8(8)2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362462

RESUMO

The primary cilium has gone from being a vestigial organelle to a crucial signaling hub of growing interest given the association between a group of human disorders, collectively known as ciliopathies, and defects in its structure or function. In recent years many ciliogenesis proteins have been observed at extraciliary sites in cells and likely perform cilium-independent functions ranging from regulation of the cytoskeleton to vesicular trafficking. Perhaps the most striking example is the non-ciliated T lymphocyte, in which components of the ciliary machinery are repurposed for the assembly and function of the immunological synapse even in the absence of a primary cilium. Furthermore, the specialization traits described at the immunological synapse are similar to those seen in the primary cilium. Here, we review common regulators and features shared by the immunological synapse and the primary cilium that document the remarkable homology between these structures.

6.
Front Cell Dev Biol ; 7: 110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31334230

RESUMO

Ligation of the T-cell antigen receptor (TCR) by cognate peptide bound to the Major Histocompatibility Complex on the surface of an antigen-presenting cell (APC) leads to the spatial reorganization of the TCR and accessory receptors to form a specialized area of intimate contact between T cell and APC, known as the immunological synapse (IS), where signals are deciphered, coordinated, and integrated to promote T cell activation. With the discovery that an endosomal TCR pool contributes to IS assembly and function by undergoing polarized recycling to the IS, recent years have witnessed a shift from a plasma membrane-centric view of the IS to the vesicular trafficking events that occur at this location following the TCR-dependent translocation of the centrosome toward the synaptic membrane. Here we will summarize our current understanding of the trafficking pathways that are responsible for the steady delivery of endosomal TCRs, kinases, and adapters to the IS to sustain signaling, as well as of the endocytic pathways responsible for signal termination. We will also discuss recent evidence highlighting a role for endosomes in sustaining TCR signaling after its internalization at the IS and identifying the IS as a site of formation and release of extracellular vesicles that allow for transcellular communication with the APC.

7.
Nat Commun ; 10(1): 2864, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253807

RESUMO

The T cell immune synapse is a site of intense vesicular trafficking. Here we show that the golgin GMAP210, known to capture vesicles and organize membrane traffic at the Golgi, is involved in the vesicular transport of LAT to the immune synapse. Upon activation, more GMAP210 interact with LAT-containing vesicles and go together with LAT to the immune synapse. Regulating LAT recruitment and LAT-dependent signaling, GMAP210 controls T cell activation. Using a rerouting and capture assay, we show that GMAP210 captures VAMP7-decorated vesicles. Overexpressing different domains of GMAP210, we also show that GMAP210 allows their specific delivery to the immune synapse by tethering LAT-vesicles to the Golgi. Finally, in a model of ectopic expression of LAT in ciliated cells, we show that GMAP210 tethering activity controls the delivery of LAT to the cilium. Hence, our results reveal a function for the golgin GMAP210 conveying specific vesicles to the immune synapse.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Golgi/fisiologia , Leucócitos Mononucleares/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/fisiologia , Linhagem Celular , Proteínas do Citoesqueleto , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Linfócitos T/fisiologia
8.
Front Immunol ; 10: 1041, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31134091

RESUMO

Multiple sclerosis is an autoimmune disease caused by autoreactive immune cell infiltration into the central nervous system leading to inflammation, demyelination, and neuronal loss. While myelin-reactive Th1 and Th17 are centrally implicated in multiple sclerosis pathogenesis, the local CNS microenvironment, which is shaped by both infiltrated immune cells and central nervous system resident cells, has emerged a key player in disease onset and progression. We have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice protects from experimental autoimmune encephalomyelitis. Here, we have explored the mechanisms that underlie the ability of Rai-/- astrocytes to antagonize T cell-dependent neuroinflammation. We show that Rai deficiency enhances the ability of astrocytes to upregulate the expression and activity of the ectonucleotidase CD39, which catalyzes the conversion of extracellular ATP to the immunosuppressive metabolite adenosine, through both contact-dependent and-independent mechanisms. As a result, Rai-deficient astrocytes acquire an enhanced ability to suppress T-cell proliferation, which involves suppression of T cell receptor signaling and upregulation of the inhibitory receptor CTLA-4. Additionally, Rai-deficient astrocytes preferentially polarize to the neuroprotective A2 phenotype. These results identify a new mechanism, to which Rai contributes to a major extent, by which astrocytes modulate the pathogenic potential of autoreactive T cells.

9.
Cancer Cell Int ; 19: 67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948927

RESUMO

Background: Low molecular weight protein tyrosine phosphatase (LMW-PTP) is overexpressed in different cancer types and its expression is related to more aggressive disease, reduced survival rate and drug resistance. Morin is a natural polyphenol which negatively modulates, among others, the activity of LMW-PTP, leading to the potentiation of the effects of different antitumoral drugs, representing a potential beneficial treatment against cancer. Methods: LMW-PTP levels were measured by immunoblot analysis both in CLL cells from patients and in chronic lymphocytic leukemia (CLL)-derived Mec-1 cells. Cell viability was assessed in Mec-1 cells treated with morin alone or in combination with either fludarabine or ibrutinib or following siRNA-mediated LMW-PTP knockdown. Furthermore, the expression levels of VLA-4 and CXCR4 were assessed by both qRT-PCR and flow cytometry and both adhesion to fibronectin-coated plates and migration toward CXCL12 were analyzed in Mec-1 cells treated with morin alone or in combination with fludarabine or ibrutinib. Results: We observed that LMW-PTP is highly expressed in Mec-1 cells as well as in leukemic B lymphocytes purified from CLL patients compared to normal B lymphocytes. Morin treatment strongly decreased LMW-PTP expression levels in Mec-1 cells and potentiated the anticancer properties of both fludarabine and ibrutinib by increasing their apoptotic effects on leukemic cells. Moreover, morin negatively regulates adhesion and CXCL12-dependent migration of Mec-1 cells by affecting VLA-4 integrin expression and CXCR4 receptor recycling. Conclusions: Morin treatment in CLL-derived Mec-1 cell line synergizes with conventional anticancer drugs currently used in CLL therapy by affecting leukemic cell viability and trafficking.

10.
Haematologica ; 104(10): 2040-2052, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30819907

RESUMO

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

11.
Cell Mol Life Sci ; 76(16): 3249-3261, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30830241

RESUMO

In addition to their modulation through de novo expression and degradation, surface levels of chemokine receptors are tuned by their ligand-dependent recycling to the plasma membrane, which ensures that engaged receptors become rapidly available for further rounds of signaling. Dysregulation of this process contributes to the pathogenesis of chronic lymphocytic leukemia (CLL) by enhancing surface expression of chemokine receptors, thereby favoring leukemic cell accumulation in the protective niche of lymphoid organs. In this review, we summarize our current understanding of the process of chemokine receptor recycling, focusing on the impact of its dysregulation in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Quimiocinas/metabolismo , Arrestinas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/metabolismo , Fosforilação , Transdução de Sinais
12.
Cell Microbiol ; 21(5): e13006, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30646431

RESUMO

Helicobacter pylori (HP) is a Gram-negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host-pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild-type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T-cell antigen receptor-dependent signalling and lymphocyte proliferation, as well as the CXCL12-dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T-cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP-associated diseases, is dependent on T cells.

14.
Autophagy ; 14(12): 2117-2138, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30109811

RESUMO

Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.


Assuntos
Linfócitos B/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/fisiologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Mitocôndrias/fisiologia , Membranas Mitocondriais/patologia , Oxidantes/metabolismo , Permeabilidade , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética
15.
Pharmacol Res ; 134: 118-133, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29898412

RESUMO

The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response.


Assuntos
Antineoplásicos/farmacologia , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Sinapses Imunológicas/efeitos dos fármacos , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Animais , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo
16.
Front Immunol ; 9: 919, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29765373

RESUMO

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the ß-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.


Assuntos
Adenilil Ciclases/metabolismo , Bacillus anthracis/enzimologia , Bordetella pertussis/enzimologia , AMP Cíclico/biossíntese , Sinapses Imunológicas/metabolismo , Linfócitos T/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Compartimento Celular/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Ativação Linfocitária , Transdução de Sinais/imunologia
17.
Front Immunol ; 9: 330, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29545793

RESUMO

Toll-like receptors (TLRs) play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF) family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs) in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1) protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their interaction with TNFR-associated factor 6 (TRAF6) and promotes the recruitment of both cIAP2 and IRF-1 by TRAF6. Collectively, our data demonstrate that TLR7/8 engagement leads to the formation of a complex that allows the interaction of cIAP2 and IRF-1 resulting in IRF-1 K63-linked ubiquitination, and that active SFKs are required for this process.


Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos B/imunologia , Fator Regulador 1 de Interferon/imunologia , Monócitos/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Ubiquitinação/efeitos dos fármacos , Quinases da Família src/imunologia , Linfócitos B/citologia , Células HEK293 , Humanos , Fator Regulador 1 de Interferon/genética , Monócitos/citologia , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genética , Ubiquitinação/genética , Ubiquitinação/imunologia , Quinases da Família src/genética
18.
Oncogene ; 37(11): 1534-1550, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29326436

RESUMO

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5+ endosomes as serine-phosphoproteins bound to ß-arrestin. This results from the ability of p66Shc to inhibit Ca2+ and PP2B-dependent CXCR4/CCR7 dephosphorylation and ß-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.


Assuntos
Endossomos/metabolismo , Leucemia Linfocítica Crônica de Células B , Receptores CCR7/metabolismo , Receptores CXCR4/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , beta-Arrestinas/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , Endossomos/efeitos dos fármacos , Endossomos/patologia , Mutação em Linhagem Germinativa , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Knockout , Fosforilação/genética , Proteólise , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico
19.
F1000Res ; 6: 1880, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29123650

RESUMO

The cell's ability to communicate with the extracellular environment, with other cells, and with itself is a crucial feature of eukaryotic organisms. In the immune system, T lymphocytes assemble a specialized structure upon contact with antigen-presenting cells bearing a peptide-major histocompatibility complex ligand, known as the immunological synapse (IS). The IS has been extensively characterized as a signaling platform essential for T-cell activation. Moreover, emerging evidence identifies the IS as a device for vesicular traffic-mediated cell-to-cell communication as well as an active release site of soluble molecules. Here, we will review recent advances in the role of vesicular trafficking in IS assembly and focused secretion of microvesicles at the synaptic area in naïve T cells and discuss the role of the IS in transcellular communication.

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