Studies from Dr. Feng Shao's laboratory discover the pore-forming activity in the gasdermin-family proteins and therefore reveal the molecular mechanism of pyroptotic cell death.

On June 8, 2016 ---- Studies from Dr. Feng Shao's laboratory discover the pore-forming activity in the gasdermin-family proteins and therefore reveal the molecular mechanism of pyroptotic cell death. The work entitled "Pore-forming activity and structural autoinhibition of the gasdermin family" is published as an "article" in the journal Nature as Advance Online Publication.

Pyroptosis is a kind of programmed necrosis manifested by cell swelling and plasma membrane lysis, resulting in release of cytosolic contents and strong inflammation. Pyroptosis plays crucial roles in innate defense against infections and endogenous dangers. Excessive pyroptosis causes many inflammatory and immunological diseases including septic shock. Traditionally, pyroptosis is known to be mediated by inflammatory caspases, caspase-1 and caspase-4/5/11. Caspase-1 is activated by pathogen signal-induced inflammasome complex; previous work from the Shao lab shows that caspase-4/5/11 are cytosolic receptors for bacterial LPS (endotoxin) and undergo oligomerization/activation upon direct binding to LPS, serving as a key determinant for Gram-negative bacteria-induced septic shock (Shi et al., Nature 2014). However, the mechanism for how inflammatory caspases trigger pyroptosis was a mystery for more than two decades. In a breakthrough study last year, the Shao lab identifies a novel GSDMD protein as a common substrate for all inflammatory caspases; caspase cleavage of GSDMD releases the N-terminal Gasdermin-N domain that by itself can drive cell pyroptosis (Shi et al., Nature 2015). GSDMD belongs to an unknown-function gasdermin family, which also includes GSDMA, GSDMB, GSDMC, DFNA5 and DFNB59. Genetic mutations in human DFNA5 and mouse Gsdma3 cause human nonsyndromic hearing impairment and mouse alopecia and skin inflammation, respectively. GSDMB polymorphism is associated with early childhood asthma. The 2015 study shows that GSDMA3 is not sensitive to inflammatory caspases but its Gasdermin-N domain can also trigger pyroptosis; alopecia-associated GSDMA3 mutants are deficient in the intramolecular autoinhibition and become constitutive in inducing pyroptosis. Despite these, the mechanism for how cleaved GSDMD triggers pyroptosis and how other gasdermins function in pyroptosis-related defense is unknown, an emerging important question    in innate immunity.

In the new study, Dr. Feng Shao and his team first found that Gasdermin-N domains from nearly all family members harbored pyroptosis-inducing activity. Except for GSDMD, other gasdermins are not substrates of inflammatory caspases. This finding re-defines the concept of pyroptosis and opens a new field in programmed necrosis. Interestingly, Gasdermin-N domains also exhibited cytotoxicity when expressed in bacteria. This hints that Gasdermin-N may have membrane-disrupting activity and therefore kill both bacteria and mammalian cells. To test this hypothesis, the researchers prepared active GSDMD, GSDMA and GSDMA3, and showed that the three gasdermin proteins could specifically bind to two membrane lipids, phosphoinositides and cardiolipin, in the liposome cosedimentation assay. Given that phosphatidylinositol-4,5-bisphosphate and cardiolipin are major lipids in mammalian cell plasma membrane and bacterial membrane, respectively, this observation correlates well the cytotoxicity of Gasdermin-N in mammalian and bacterial cells. Further biochemical and immunofluorescence imaging analyses showed that activated Gasdermin-N domain moved from the cytosol to plasma membrane during pyroptosis, following which cell membrane bubbles appeared accompanied by cell swelling. Moreover, recombinant Gasdermin-N could only kill mammalian cells from the inside and showed no membrane lysis activity when added extracellularly, which is perfectly consistent with that phosphoinositides are only present in the cytoplasmic leaflet of plasma membrane.

Using the liposome-leakage assay, the Shao team further found that Gasdermin-N could efficiently and specifically induce leakage of phosphoinositide- or cardiolipin-containing liposomes. When liposomes encapsulated with different-size dextrans were assayed, it was noted that items with diameters of 10 nm or less could pass through the broken liposome, suggesting that Gasdermin-N may form regular membrane pores. Biochemical crosslinking assay confirmed the oligomerization of Gasdermin-N upon liposome binding or translocation onto the plasma membrane during pyroptosis. The researchers then employed negative-stain electron microscopy and discovered that gasdermin-N domains formed multiple pores of "Swiss cheese"-like shape on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10- 14 nm. In-depth electron microscopy analyses revealed a 16-fold symmetry, suggesting a 16-mer symmetric pore complex on the membrane formed by the Gasdermin-N domain. The researchers also determined the 1.90 Å crystal structure of GSDMA3, which revealed unique structural feature of gasdermin-N as a new type of pore-forming protein as well as the detailed autoinhibitory interactions. Structure-guided point-mutation analyses further confirmed that the liposome-binding and pore-forming activities of the gasdermin-N domain are the molecular basis for the pyroptosis-inducing activity.

The study has proved that GSDMD protein is the executioner in inflammatory caspase-mediated pyroptosis, and importantly for the first time reveals that the gasdermin family has a pore-forming membrane-disrupting activity. These findings not only establish the molecular mechanism of inflammatory caspases-mediated pyroptosis but also re-define the concept of pyroptosis as gasdermin-mediated programmed necrosis. The study should guide GSDMD-based drug development for inflammatory caspases-associated autoinflammatory diseases as well as septic shock and also paves the way for future studies to elucidate the functional mechanism of other gasdermin proteins.

Visiting scientist Dr. Jingjin Ding from Professor Da-Cheng Wang's group at Institute of Biophysics (CAS) is the first author of this paper; PhD student Kun Wang in the Shao laboratory also made important contributions. Other authors of the paper include PhD students Wang Liu, Yang She and Qi Sun as well as postdoc fellows Jianjin Shi and Hanzi Sun. Drs. Da-Cheng Wang and Feng Shao are the co-corresponding authors. The research was supported by the 973 National High-Tech. Projects, the Beijing Municipal Government, China National Science Foundation, the Strategic Priority Research Program of the Chinese Academy of Sciences and Howard Hughes Medical Institute in the States, and carried out at National Institute of Biological Sciences, Beijing.

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature18590.html