Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments (CCFs) in senescent cells. The activation of cGAS, in turn triggers the production of SASP factors via Stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence upon irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

histones in situ, which were partially lost upon aclarubicin treatment (Extended Data Fig. 1c, d). Thus, histones appear to dynamically engage cGAS in the nucleus.Consistent with prior work [13], functional analysis of cGAS in vitro enzymatic activity revealed that mononucleosomes (hereafter nucleosomes) inhibited DNA-induced cGAMP synthesis (Extended Data Fig. 1e). Likewise, compact chromatin fibres (12-mer nucleosome arrays) suppressed cGAS activity (Extended Data Fig. 1e). H2A-H2B dimers also had an inhibitory effect, but neither H2A or H2B monomers nor H3 or H4 monomers, respectively (Extended Data Fig. 1f, g). Thus, H2A-H2B dimers on their own can suppress cGAS (Extended Data Fig. 1h), albeit with weaker overall potency compared to fullassembled nucleosomes with additional features of nucleosomes in chromatin being necessary to exert maximal inhibition. Overall structure of the cGAS-NCP complexTo determine how cGAS interacts with nucleosomes, we pursued structural studies. A 1.5:1 molar mixture of human cGAS (residues 155 to 522) with a 147 bp 601 DNA nucleosome core particle (NCP) resulted in heterogenous particle distributions (Extended Data Fig. 2ad). To select for and stabilize more homogenous cGAS-NCP complexes, we combined gradient centrifugation with chemical crosslinking (GraFix) [15]. Both WT cGAS and cGAS K394E, a mutant impaired in dsDNA-mediated cGAS dimerisation [16], were used for structure determination. For the cGAS K394E mutant, we obtained a 4.1 Å reconstruction revealing two NCPs organized in a NCP 1 -cGAS 1 -cGAS 2 -NCP 2 sandwich arrangement with an expected molecular weight around 560 kDa, consistent with the most prominent peak fraction in multi-angle light scattering (MALS) (Fig. 1a, b, Extended Data Fig. 3, Supplementary Video 1, 2, and Extended Data Table 1a). The two individual nucleosomes are held together by two cGAS protomers. While the first cGAS protomer and its corresponding NCP (designated cGAS 1 and NCP 1 ) are well-resolved, the second nucleosome/cGAS pair (NCP 2 and cGAS 2 ) is less ordered (Extended Data Fig. 3e). In the dimeric NCP 1 -cGAS 1 -cGAS 2 -NCP 2 arrangement, each cGAS protomer interacts with the histone octamer of one NCP through histones H2A and H2B and the nucleosomal DNA (e.g. cGAS 1 and NCP 1 ), while contacting the second nucleosome (e.g. cGAS 1 and NCP 2 ) primarily through interactions with the nucleosomal DNA (Fig. 1a, b). In the WT cGAS structure, we observed a similar overall structural arrangement, with the NCP 1 -cGAS 1 -cGAS 2 -NCP 2 complex at 5.1Å and the focused NCP 1 -cGAS 1 structure at 4.7Å resolution (Extended Data

The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.

Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11 −/− cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.interleukin-1beta | pyroptosis | lethal toxin | macrophage | dendritic cell

NFκB is a master regulator of protein quality control. It helps the cells to survive proteotoxicity by modulating autophagy via up-regulation of BAG3 and HspB8 expression, a molecular mechanism relevant to protein conformational diseases.

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The caspase-1 enzymatic activity plays a major role in the innate immune response as it regulates the maturation of two major proinflammatory cytokines, the interleukin-1beta (IL-1β) and IL-18. In this chapter, we describe the technique of Western blot to assess caspase-1 activation. This method provides multiple information within one experiment. It allows the detection of both unprocessed and processed caspase-1 and substrates.