Study On The Mechanism Of Pyrin Inflammasome Activation And Gasdermin E Induced Pyroptosis

Pyrin encoded by MEFV is well known for its gain-of-function mutations causing familiar mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a Caspase-1 -activating inflammasome in response to inactivated Rho GTPases modified by various bacterial toxins or effectors.Pyrin-mediated innate immunity is unique because it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Using Western blot, we found that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. The phosphorylation of Ser205 and Ser241 resulted in inhibitory binding by cellular 14-3-3 proteins. These 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 Ser205 and Ser241, which confirmed stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses of Ser205/241 indicated that both phosphorylation and signal-induced dephosphorylation are important for Pyrin activation. Moreover, microtubule drugs,including Colchicines commonly used in clinical treatment of FMF, effectively blocked Pyrin inflammasome activation. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated ASC aggregation. Our study reveals that site-specific phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity.Pyroptosis, caused by activated Caspase-1 and Caspase-4/5/11, is critical for immune defenses and various diseases including sepsis. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity. GSDMD belongs to a Gasdermin family sharing the autoinhibited pore-forming domain.However, the physiological function and mechanism of activation of other Gasdermins are unknown.Using Western blot and image detection we found that expression of Gasdermin E (GSDME) could switch TNF-induced Caspase-3-mediated apoptosis to pyroptosis. Similar to Caspase-1/11/4/5 cleavage and activation of GSDMD, Caspase-3 cleavage released the autoinhibition in GSDME, generating an active fragment that could break cell membrane and trigger cell pyroptosis. Caspase-3-mediated GSDME activation determined pyroptosis in SH-SY5Y cell upon treatment with various chemotherapy drugs. In GSDME-positive primary cells derived from normal human tissues, pyroptotic death occurred upon caspase-3 activation by chemotherapy drugs. Blocking of caspase-3 activation or knockdown of GSDME expression both inhibited the drugs-induced pyroptosis in the primary cells. Gsdme-/- mice were protected from DNA damaging chemotherapy-induced tissue damage of lung and small intestine.These findings suggest that Caspase-3 activation can trigger inflammatory necrosis by cleaving GSDME and the expression of GSDME becomes the control element of the way to death of the cell.