Vind, A. C. | Search bioRxiv

bioRxiv2025

ZAKα is a sensor of mRNA stasis at the ribosomal exit channel

Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38 and JNK-activating MAP3 kinase ZAK senses translational impairment. Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAK dynamically monitors the mRNA exit channel of elongating ribosomes for mRNA stasis. This is accomplished by ZAK via its direct interactions with the ribosomal proteins RACK1 and RPS27 as well as with the 18S rRNA helix-26. In this conformation, four mRNA-binding peptides in ZAK span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAK off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAK to slow-elongating, stalled and collided ribosomes is associated with sequestration of the inhibitory SAM domain on RACK1, allowing for transient ZAK dimerization, activation loop trans-autophosphorylation and RSR activation. We propose that compromised ribosome processivity constitutes a common ribotoxic stress signal and that ZAK is a ribosome collision-agnostic sensor of such perturbations.

molecular biology↗

bioRxiv2023

Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple non-hematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAK[a], p38 and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAK[a] knockout or pharmacologic inhibitors of ZAK[a] and p38 kinase activities. By surveying a diverse panel of ionophores, we show that the electroneutrality of potassium efflux is essential to activate ZAK[a]-driven RSR, likely because a greater extent of K+ depletion is necessary to activate ZAK[a]-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux and innate immunity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/546021v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1afe96aorg.highwire.dtl.DTLVardef@53c59org.highwire.dtl.DTLVardef@1bd14d1org.highwire.dtl.DTLVardef@a1bccc_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCENigericin is familiar to the inflammasome field as the most robust and commonly used NLRP3 inducer. It has enabled numerous breakthroughs in the field linking NLRP3 activation to potassium efflux. In this manuscript, we report that nigericin activates an alternate inflammasome sensor, NLRP1 in primary human skin, nasal and corneal epithelial cells. NLRP1 activation by nigericin requires K+ efflux-driven ribosome stalling and the ribotoxic stress response (RSR) sensor MAP3K, ZAK[a]. We further identify the key biophysical principles that explain why only a subset of K+ ionophores, exemplified by nigericin, function as super inflammasome agonists that can activate either NLRP1 or NLRP3, depending on cell type. These results reveal an unexpected connection between RSR, potassium ion flux and innate immunity.

immunology↗