Hepatitis C virus (HCV) entry is a critical step in establishing infection and is highly dependent on host cell co-receptors. We previously identified that the tight junction protein CLDN12 facilitates HCV entry. However, the regulatory mechanisms governing the dynamic post-translational modifications of CLDN12 remain unclear. This study employs targeted mutagenesis, palmitoylation level analysis, plasma membrane localization, manipulation of key enzymes, and functional virology assays to reveal whether and how reversible palmitoylation regulates the function of CLDN12 in HCV entry. We found that the palmitoylation occurs at cysteine residues C3 and C109 of CLDN12, a modification critical for its role in mediating HCV entry. The palmitoyltransferase ZDHHC7 is a key enzyme that catalyzes the palmitoylation of CLDN12, positively promoting HCV entry. Acyl-protein thioesterases APT1 and APT2 regulate the de-palmitoylation of CLDN12 through differential modulation at the two sites, thereby negatively regulating HCV entry. Our study discovers that ZDHHC7 and APT1/2 form a reversible palmitoylation switch, which regulates the efficiency of HCV entry by dynamically controlling the plasma membrane localization of CLDN12. This finding reveals a new mechanism by which HCV exploits the host’s palmitoylation cycle to promote infection, and highlights this regulatory axis as a potential target for antiviral intervention.
NLRP3 inflammasome is activated by diverse stimuli including infections, intracellular and environmental irritants. How NLRP3 senses these unrelated stimuli and what activates NLRP3 remain unknown. Here we report that signal-dependent NLRP3 phase separation initiated its activation, in which the palmitoyltransferase ZDHHC7-mediated tonic NLRP3 palmitoylation and an IDR region in the FISNA domain of NLRP3 play important roles. Moreover, three conserved hydrophobic residues in the IDR critically mediate multivalent weak interactions. NLRP3-activating stimuli including K + efflux and NLRP3-interacting molecules imiquimod, palmitate, and cardiolipin all cause NLRP3 conformational change and induce its phase separation and activation in cells and/or in vitro. Surprisingly, amphiphilic molecules like di-alcohols used to inhibit biomolecular phase separation and chemotherapeutic drugs doxorubicin and paclitaxel activate NLRP3 independently of ZDHHC7 by directly inducing NLRP3 phase separation. Mechanistically, amphiphilic molecules decrease the solubility of both palmitoylated and non-palmitoylated NLRP3 to directly induce its phase separation and activation while NLRP3 palmitoylation reduces its solubility to some extent without activation. Therefore, ZDHHC7-mediated NLRP3 palmitoylation in resting cells licenses its activation by lowering the threshold for NLRP3 phase separation in response to any of the diverse stimuli whereas NLRP3 solubility-reducing molecules like di-alcohols and chemotherapeutic drugs activate NLRP3 directly. The signal-induced NLRP3 phase separation likely provides the simplest and most direct mechanistic basis for NLRP3 activation.
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