ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia.

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131 This cleavage triggers N-glycine-mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.

Cutaneous SCC (cSCC) is the most frequently occuring skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors. Here, we use massively parallel exome and targeted level sequencing of 132 sporadic cSCCs and of 39 squamoproliferative lesions and cSCCs arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples, to identify NOTCH1 mutation as an early event in squamous cell carcinogenesis. Bisected vemurafenib-induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC, suggesting polyclonal origin. Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCCs, and regions of NOTCH1 loss or downregulation are frequently observed in normal-looking skin. Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.

Diazido mixed-amine platinum(IV) anticancer complexes activatable by visible-light form novel DNA adducts.

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side-effects. Photoactivatable Pt(IV) prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X-ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (1; MA=methylamine, Py=pyridine) and trans,trans,trans-[Pt(N3)2(OH)2(MA)(Tz)] (2; Tz=thiazole), and interpret their photophysical properties by TD-DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1p and 1q. Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin-resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf-thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono- and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross-links, with evidence for DNA strand cross-linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin-type lesions. The photo-induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt(II) compounds trans-[PtCl2(MA)(Py)] (5) and trans-[PtCl2(MA)(Tz)] (6). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1, whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.