Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

Apoptosis-induced tolerance. Role of the novel cytokine IL-38

Introduction: Necrotic cell death triggers inflammation, whereas apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction and are produced by necrotic cells to induce sterile inflammation. Release of IL-1 family proteins from apoptotic cells to regulate inflammation has not been described. The novel cytokine IL-38 shares homology with IL-1 family receptor antagonists and was therefore proposed as a negative regulator of IL-1 family receptor signaling. Methods: IL-38 concentrations were measured by ELISA. Apoptotictumor cell conditioned medium (ACM) was used to stimulate human primary immune cells. The response to ACM was determined as cytokine release (Cytometric Bead Array) as well as transcription factor activity (Luciferase reporter assays). N-terminal processing of IL-38 was determined by mass spectrometry. Binding of IL-38 to its putative receptors was determined by receptor binding assays. The pathophysiological function of IL-38 was analyzed using the imiquimod (IMQ)-induced psoriasis mouse model in IL-38-deficient animals compared to WT controls. Statistical analysis was performed using ANOVA with Bonferroni’s correction. Results: We show that IL-38 is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing IL-17-producing T cell activation. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1). However, we show higher affinity binding of mature IL-38. Likewise, we confirmed the previously reported low-affinity binding of the IL-38 precursor to IL1R1 and show an increased affinity of mature IL-38 to this receptor. Functionally, the IL-38 precursor induced an increase in IL-6 production by human macrophages, whereas truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. Ongoing studies show selective secretion of IL-38 from dying cells challenged with tolerogenic chemotherapy, but not with immunogenic chemotherapy. Moreover, strengthening the role of IL-38 as a tolerogenic factor, IL-38-deficient mice subjected to IMQ-induced psoriasis show an increased IL-17-mediated immune response and a strong delay in the restoration of skin architecture. Conclusion: We identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion. IL-38 limits cytokine production in macrophages antagonizing the IL1-RAPL1/JNK/AP1 pathway and subsequently preserving a low T cell IL-17 production. We propose apoptotic cell-derived IL-38 as the counter-regulatory equivalent of necrotic cell-derived alarmins of the IL-1 family, which might be relevant in resolution of inflammation, autoimmunity, and cancer

PLCbeta isoforms differ in their subcellular location and their CT-domain dependent interaction with Galphaq

Item does not contain fulltextPhospholipase C (PLC) beta isoforms are implicated in various physiological processes and pathologies. However, mechanistic insight into the localization and activation of each of the isoforms is limited. Therefore, it is crucial to gain more in-depth knowledge as to the regulation of the different isoforms. Here we describe the subcellular location of full-length PLCbeta isozymes and their C-terminal (CT) domains. Strikingly, we found isoforms PLCbeta1 and PLCbeta4 to be enriched at the plasma membrane, contrary to isoforms PLCbeta2 and PLCbeta3. We determined that the CT domain is an inhibitor of Gq-mediated increases in intracellular calcium, the potency of its effect being dependent upon the CT domain isoform used. Furthermore, ratiometric fluorescence resonance energy transfer (FRET) imaging was used to study the kinetics of the Galphaq-CTbetax interactions. By the use of recently developed tools, which enable the on-demand activation of Galphaq, we could show that the interaction between constitutively active Galphaq and PLCbeta3 prolongs the residence time of PLCbeta3 at the plasma membrane. These findings suggest that under physiological circumstances, PLCbeta3 and Galphaq interact in a kiss-and-run fashion, likely due to the GTPase-activating activity of PLCbeta towards Galphaq

Interleukin-38 is released from apoptotic cells to limit inflammatory macrophage responses

Different modes of cell death regulate immunity. Whereas necrotic (necroptotic, pyroptotic) cell death triggers inflammation, apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction. A number of IL-1 family cytokines are produced by necrotic cells to induce sterile inflammation. However, release of IL-1 family proteins from apoptotic cells to regulate inflammation was not described. Here we show that IL-38, a poorly characterized IL-1 family cytokine, is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing Th17 cell expansion at the expense of IL-10-producing T cells. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin -1 receptor accessory protein-like 1 (IL1RAPL1). However, whereas the IL-38 precursor induced an increase in IL-6 production by human macrophages, truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. In conclusion, we identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion, which might be relevant in resolution of inflammation, autoimmunity, and cancer. © The Author (2016)

Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins

Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin‐like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3‐interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C‐specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs