Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

Abstract

Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity.

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation

“…S1 G-K). Thus, our results confirm recent findings that purified TcdA selectively engages the Pyrin inflammasome in mouse BMDMs (19) and extend these results to human PBMCs.…”

“…Dephosphorylation of Pyrin's intermediate linker domain was recently shown to be required for TcdA-and TcdB-induced inflammasome activation (14,15,19), and introduction of FMF mutations in the B30.2 domain of ectopically expressed Pyrin did not interfere with this process (15). In agreement with these reports, we showed that endogenous Pyrin was not constitutively active in FMF PBMCs but was engaged only after TcdA stimulation or C. difficile infection ( Fig.…”

“…3D). As reported (19), TcdA also triggered Pyrin dephosphorylation in wild-type BMDMs that had been pretreated with colchicine (Fig. 3E).…”

Genomics, Biology, and Human Illness

“…Park et al demonstrated 76 that colchicine reverses the effect of RhoA inhibition by C3 toxin from Clostridium botulinum , and furthermore, suppresses the spontaneous activation of the pyrin inflammasome in FMF knockin mice and in FMF patients’ cells, but not in the cells from CAPS patients. In addition, Feng Shao and colleagues 84 demonstrated that multiple microtubule-targeting drugs including colchicine, vinblastine, and paclitaxel inhibit pyrin inflammasome-mediated ASC aggregation. These data are consistent with the clinical response of FMF patients to colchicine treatment and the exquisite specificity of colchicine for FMF among the hereditary periodic fever syndromes.…”

Lessons from characterization and treatment of the autoinflammatory syndromes

“…Although there is some uncertainty regarding the precise details, there is an emerging consensus that colchicine is a specific inhibitor of the pyrin inflammasome [38,41]. Taken together, these very recent studies on the regulation of pyrin inflammasome activity have answered many questions related to genotype-phenotype correlations, pathophysiology and efficacy of treatment in patients with pyrin-associated conditions.…”