RESUMO
Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.
Assuntos
COVID-19 , Monócitos , Humanos , SARS-CoV-2 , Proteômica , Macrófagos , Fatores de TranscriçãoRESUMO
Although homo- and heterodimerization are reported for some chemokine receptors, it remains unclear whether these functional states are in dynamic equilibrium and how receptor/ligand levels influence oligomerization. In human neutrophils and in cell lines that coexpress the chemokine receptors CXCR1 and CXCR2, we used fluorescence resonance energy transfer techniques to show that these two receptors form homo- and heterodimers. Receptor expression and ligand activation were found to regulate the balance between these complexes, adapting the response to changes in the milieu. CXCL8, a ligand for both receptors, alters heterodimeric complexes, whereas it stabilizes homodimers and promotes receptor internalization. Oligomerization of receptors, together with the regulation of their expression and desensitization, could thus contribute to the fine control of chemokine functions.
Assuntos
Neutrófilos/imunologia , Receptores de Interleucina-8A/química , Receptores de Interleucina-8B/química , Western Blotting , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Interleucina-8/química , Interleucina-8/imunologia , Interleucina-8/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , TransfecçãoRESUMO
The limited availability of liver donors and recent progress in cell therapy technologies has centered interest on cell transplantation as a therapeutic alternative to orthotopic liver transplant for restoring liver function. Following transplant by intraportal perfusion, the main obstacle to cell integration in the parenchyma is the endothelial barrier. Transplanted cells form emboli in the portal branches, inducing ischemia and reperfusion injury, which cause disruption of endothelial impermeability and activate the immune system. Approximately 95% of transplanted cells fail to implant and die within hours by anoikis or are destroyed by the host immune system. Intravascular perfusion of Bordetella pertussis toxin (PTx) blocks endothelial G(i) proteins and acts as a reversible inducer of actin cytoskeleton reorganization, leading to interruption of cell confluence in vitro and increased vascular permeability in vivo. PTx treatment of the murine portal vascular tree 2 h before intraportal perfusion of embryonic stem cells facilitated rapid cell engraftment. By 2 h postperfusion, the number of implanted cells in treated mice was more than fivefold greater than in untreated controls, a difference that was maintained to at least 30 days posttransplant. We conclude that prior to cell transplant, PTx blockade of the G(i) protein pathway in liver endothelium promotes rapid, efficient cell implantation in liver parenchyma, and blocks chemokine receptor signaling, an essential step in early activation of the immune system.
Assuntos
Células-Tronco Embrionárias/citologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Fígado/citologia , Citoesqueleto de Actina , Animais , Células Cultivadas , Células-Tronco Embrionárias/transplante , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Toxina Pertussis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Cytosolic division in mitotic cells involves the function of a number of cytoskeletal proteins, whose coordination in the spatio-temporal control of cytokinesis is poorly defined. We studied the role of p85/p110 phosphoinositide kinase (PI3K) in mammalian cytokinesis. Deletion of the p85alpha regulatory subunit induced cell accumulation in telophase and appearance of binucleated cells, whereas inhibition of PI3K activity did not affect cytokinesis. Moreover, reconstitution of p85alpha-deficient cells with a Deltap85alpha mutant, which does not bind the catalytic subunit, corrected the cytokinesis defects of p85alpha(-/-) cells. We analyzed the mechanism by which p85alpha regulates cytokinesis; p85alpha deletion reduced Cdc42 activation in the cleavage furrow and septin 2 accumulation at this site. As Cdc42 deletion also triggered septin 2 and cytokinesis defects, a mechanism by which p85 controls cytokinesis is by regulating the local activation of Cdc42 in the cleavage furrow and in turn septin 2 localization. We show that p85 acts as a scaffold to bind Cdc42 and septin 2 simultaneously. p85 is thus involved in the spatial control of cytosolic division through regulation of Cdc42 and septin 2, in a PI3K-activity independent manner.