RESUMO
CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.
Assuntos
Sinalização do Cálcio/imunologia , Sinapses Imunológicas/metabolismo , Proteína ORAI1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunidade Adaptativa , Proteína 1 Homóloga a Discs-Large/antagonistas & inibidores , Proteína 1 Homóloga a Discs-Large/genética , Proteína 1 Homóloga a Discs-Large/metabolismo , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Sinapses Imunológicas/genética , Sinapses Imunológicas/imunologia , Células Jurkat , Cinética , Ativação Linfocitária , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in MÏs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Proteína 1 Homóloga a Discs-Large/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Imunidade Adaptativa , Antígenos CD/biossíntese , Antígenos CD/genética , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Proteína 1 Homóloga a Discs-Large/antagonistas & inibidores , Proteína 1 Homóloga a Discs-Large/genética , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Interleucina-12/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Densidade Pós-Sináptica/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Antígeno CD83RESUMO
BACKGROUND: Benign thyroid nodules (BTN) are frequently diagnosed as papillary thyroid carcinoma (PTC), leading to unnecessary treatment. We found that plasma lncRNA DLG1-AS1 was upregulated in PTC patients but not in BTN patients and healthy controls. METHODS: In this study DLG1-AS1 and miR-199a-3p in plasma of both PTC patients and BTN patients were detected by qPCR. ROC curve analysis was performed for diagnostic analysis. Overexpression experiments were performed to analyze the interaction between DLG1-AS1 and miR-199a-3p. CCK-8 assay was performed to analyze cell proliferation. RESULTS: In this study, upregulation of DLG1-AS1 distinguished PTC patients from BTN patients and healthy controls. Plasma miR-199a-3p was downregulated in PTC patients compared with healthy controls and BTN patients. Plasma levels of miR-199a-3p were inversely correlated in PTC patients, but not in BTN patients and healthy controls. miR-199a-3p overexpression failed to significantly affect DLG1-AS1, while DLG1-AS1 overexpression resulted in downregulated miR-199a-3p, In addition, DLG1-AS1 overexpression promoted the proliferation of PTC cells. miR-199a-3p overexpression played an opposite role and attenuated the effects of DLG1-AS1 overexpression. CONCLUSIONS: Therefore, DLG1-AS1 may promote PTC by downregulating miR-199a-3p.