Cell death and inflammation in skin homeostasis and small cell lung cancer

Z-DNA binding protein 1 (ZBP1) has evolved as a key player in viral infections and tissue homeostasis promoting necroptotic cell death. Whilst a RHIM-RHIM dependent interaction between ZBP1 and RIPK1 has been implicated to exert inhibitory functions, RHIM-mediated recruitment of RIPK3 to ZBP1 was shown to induce necroptosis. In this work, we firstly show that TNFR1 and ZBP1 act synergistically to trigger skin inflammation upon keratinocyte-specific loss of FADD, reinforcing ZBP1 as a crucial player in skin homeostasis. We then aimed to shed light on the mechanisms of ZBP1 downstream signaling in presence of RIPK1 and FADD, as most studies thus far dissecting ZBP1 signaling in vivo required loss of one of these two proteins. To this aim we generated a constitutively active C-terminally truncated version of ZBP1 (ZBP1ca). Keratinocyte-specific ZBP1ca expression was able to induce apoptosis as well as necroptosis dependent on RIPK1 and RIPK3 RHIM-RHIM interactions, respectively. Mechanistically, this RIPK1-mediated apoptosis appeared to be independent of its kinase activity, identifying a novel mechanism of ZBP1-driven cell death. Having established ZBP1ca as a potent inducer of inflammation, we aimed to make use of this construct in a therapeutic setting. To this end, we utilized a mouse model for small cell lung cancer (SCLC), the most aggressive lung cancer subtype, which is in urgent need for novel targeted therapies. Our data suggest resistance of a subset of SCLC cell lines to the induction of immunogenic cell death by MLKL activation, further warranting the close investigation of molecular mechanisms underlying this deadly disease. Using different genetically modified mouse models investigating the role of inflammatory signaling in SCLC, we observed a strong dependency on NEMO/RelA-dependent NF-kB signaling for SCLC development, suggesting inhibition of NF-kB signaling as a therapeutic option for SCLC. Taken together, our study highlights a novel mechanism for ZBP1-induced cell death in sterile conditions and gives new insights about ZBP1-mediated signaling. Furthermore, we present data which can aid in the treatment of SCLC as human patients may benefit from NF-kB inhibition

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants