RESUMO
Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.
Assuntos
Endossomos/metabolismo , Interferon Tipo I/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Endossomos/ultraestrutura , Humanos , Transporte Proteico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.
Assuntos
Citosol/química , Complexos Multiproteicos/química , Análise de Célula Única , Citosol/metabolismo , Humanos , Microscopia de Fluorescência , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Propriedades de SuperfícieRESUMO
Triggered immobilization of proteins in the plasma membrane of living cells into functional micropatterns is established by using an adaptor protein, which is comprised of an antiGFP nanobody fused to the HaloTag protein. Efficient in situ reorganization of the type I interferon receptor subunits as well as intact, fully functional signaling complexes in living cells are achieved by this method.
Assuntos
Membrana Celular/metabolismo , Transdução de Sinais , Sobrevivência Celular , Células HeLa , Humanos , Proteínas Imobilizadas/metabolismo , Proteínas de Membrana/metabolismo , Microtecnologia , Receptores de Superfície Celular/metabolismoRESUMO
Bioinspired surface topographies showing generic anti-adhesive behaviour by minimization of the real contact area not only with rigid, but also with soft and compliant counterpart surfaces recently attracted increasing attention. In the present study, we show that such generic anti-adhesive surfaces, which moreover demonstrate anti-fouling behaviour, can be produced on a large scale by a simple double replication of monolayers of microspheres with diameters of a few 10 µm. Thus, we obtained mechanically stable monolithic arrays of microspheres tightly connected to a support of the same material. Adhesion of these microsphere arrays to sticky and compliant counterpart surfaces was one order of magnitude weaker than that of flat control samples of the same material. The generation of nanorod arrays with nanorod diameters of a few 100 nm as the second hierarchical structure level on monolithic microsphere arrays did not significantly affect the adhesion force. The experimental data on anti-adhesive behaviour were modelled using a modified Johnson-Kendall-Roberts theoretical approach that also provided general design criteria for topographic adhesion minimization to sticky counterpart surfaces.
Assuntos
Materiais Biomiméticos/química , Teste de Materiais , Microesferas , Modelos Químicos , Nanosferas/química , Poliestirenos/química , Adesividade , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Propriedades de SuperfícieRESUMO
ISG15 is an interferon (IFN)-α/ß-induced ubiquitin-like protein. It exists as a free molecule, intracellularly and extracellularly, and conjugated to target proteins. Studies in mice have demonstrated a role for Isg15 in antiviral immunity. By contrast, human ISG15 was shown to have critical immune functions, but not in antiviral immunity. Namely, free extracellular ISG15 is crucial in IFN-γ-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18-mediated downregulation of IFN-α/ß signalling. Here we describe ISG15-deficient patients who display no enhanced susceptibility to viruses in vivo, in stark contrast to Isg15-deficient mice. Furthermore, fibroblasts derived from ISG15-deficient patients display enhanced antiviral protection, and expression of ISG15 attenuates viral resistance to WT control levels. The species-specific gain-of-function in antiviral immunity observed in ISG15 deficiency is explained by the requirement of ISG15 to sustain USP18 levels in humans, a mechanism not operating in mice.