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Studies from Dr. Feng Shao's laboratory reveal the molecular mechanism of pyroptosis executioner GSDMD recognition by activated caspases in innate immunity

Publication Date:2020/02/28

On Feb 27, 2020 ---- Studies from Dr. Feng Shao's laboratory reveal the molecular mechanism of pyroptosis executioner GSDMD recognition by activated caspases in innate immunity. The work entitled "Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis" is published in the journal Cell.



Pyroptosis is one of the most important innate defenses against infections and endogenous dangers. Excessive pyroptosis causes inflammatory diseases such as sepsis. In a breakthrough study of 2015, the Shao lab identified a novel GSDMD protein as a common substrate for activated capase-1 and caspase-4/5/11 in innate immunity; cleavage of GSDMD by caspase induces pyroptosis (Shi et al., Nature 2015). Following this work, the Shao lab proved that GSDMD is the pyroptosis executioner; activated caspases cleave the linking loop between the N- and C-terminal domain of GSDMD, thereby unlocking the autoinhibition on the GSDMD-N domain which then translocates to plasma membrane through binding to membrane lipids and perforates the membrane to cause pyroptosis (Ding et al., Nature 2016). Despite these progress, the mechanism for how caspases in innate immunity, especially caspase-4/5/11, are activated by upstream signals, and how activated caspases specifically target GSDMD to trigger pyroptosis is unknown. This is an emerging important question in pyroptosis studies.

In this study, Dr. Feng Shao's laboratory first collaborated with Abcam, and developed two cleavage-specific anti-GSDMD antibodies which recognize the newly exposed C terminus of GSDMD-N domain and N terminus of mouse GSDMD-C domain, respectively. Both antibodies show high sensitivity and specificity to their antigens in the immunoblotting, immunofluorescence and immunocytochemistry staining assays. Using the cleavage-specific anti-GSDMD-N antibody, researchers observed a robust immunohistochemistry signal in liver samples derived from cirrhosis or liver failure patients, but not control healthy donors. Thus, the cleavage-specific anti-GSDMD antibodies can serve as a valuable tool to detect GSDMD-cleavage-mediated pyroptosis under diverse physiological and pathological contexts.

Both caspase-1 and caspase-4/11 belong to a large conserved family of cysteine proteases, named caspase. The caspase family has attracted decades of heated studies due to their close association with apoptosis. All caspases are synthesized as inactive zymogens containing a conserved protease domain divided into a large p20 and a small p10 subunit; their activation requires cleavage between the p20 and the p10 which generate a p20/p10 heterodimer. Autoprocessing of caspase-1, which can be detected by routine immunoblotting, has been a useful marker for assaying canonical inflammasome activation in innate immunity. While autoprocessing was not consistently observed in initial studies on the LPS receptors caspase-4/11. Two recent reports indicate that caspase-11 is autoprocessed between the p20 and the p10, upon cytosolic LPS stimulation, to cleave GSDMD and induce pyroptosis. However, autoprocessing of human caspase-4/5, and the connection between autoproteolysis-dependent caspase activation and GSDMD-cleavage-mediated pyroptosis remain unclear. Using caspase-4 recombinant protein purified from insect cells and a specific anti-caspase-4 antibody that recognize the N-terminal prodomain, researchers discovered that LPS-stimulated caspase-4 also undergoes autoprocessing in vitro and in treated cells. Unlike caspase-11 which autoprocesses after Asp285 at the N-terminus of p10, caspase-4 autocleaves at two sites, Asp270 at the C-terminus of p20 and Asp289 (corresponding to Asp285 in caspase-11) at the N-terminus of p10. Mutagenesis analyses further confirmed that Asp289/285 in caspase-4/11 is the only autoprocessing site critical for their activation by cytosolic LPS for cleaving GSDMD and inducing pyroptosis.

The dogma in caspase biochemistry is that all caspases recognize a tetrapeptide motif xxxD (D for aspartate, x for any residue) within a substrate and cleave after the aspartate. Extensive peptide substrate profiling has established the tetrapeptide sequence preferred by each caspase. Caspases in apoptosis often cleave a dozen of substrates. However, caspase-4/11 only have one known physiological substrate GSDMD, and caspase-1 cleave two more cytokines IL-1β/18 besides GSDMD. The caspase dogma cannot explain why caspases in innate immunity bear such a narrow substrate spectrum. Surprisingly, researchers found that mutation of the cleavage-site tetrapeptide in GSDMD did not affect caspase cleavage and pyroptosis induction. Further mapping by domain truncation and building chimeric proteins revealed that the GSDMD-C domain, but not the cleavage-site tetrapeptide mediates recognition of GSDMD by activated caspase-4/11. Researchers successfully determined the crystal structures of the complexes between autoprocessed caspase-4/11 and GSDMD-C domain. The complex structures show that autoprocessed caspase-4/11 p20/p10 further dimerize to form a heterotetramer. At the dimerization interface, the C-terminal loop of p20 in a p20/p10 bundles with the N-terminal loop of p10 in the other p20/p10, forming a two-strand intermolecular β-sheet which extends outwards, distantly from the catalytic site cysteine. The intermolecular β-sheet shapes like a key inserted into a preformed hydrophobic groove in the GSDMD-C domain. Each caspase-4/11 heterotetramer contains two symmetric intermolecular β-sheets which mediate binding of two GSDMD-C molecules, resulting in a 2:2 enzyme-substrate complex. The caspase-4/11-GSDMD complex structures also demonstrate that N-terminal extension of p10 would introduce inappropriate structural clashes to GSDMD-C domain, explaining why caspase-4/11 need to be precisely processed at Asp289/285. Mutagenesis analyses confirmed the hydrophobic interface is the structural basis for caspase-GSDMD recognition and GSDMD-cleavage-mediated pyroptosis. Researchers further investigated the caspase-1 recognition of GSDMD. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode as caspase-4/11, which is mediated by the hydrophobic interface and independently of the cleavage-site tetrapeptide.

This study provides complete and thorough understanding of caspase-1/4/11 autoprocessing and recognition of pyroptosis executioner GSDMD in innate immunity. The reported caspase-GSDMD complex structures are the first structures of enzyme-substrate complex in caspase studies. The findings of this study provide in-depth insights into substrate recognition and enzymatic mechanism of caspase family. Caspases have important functions in cancer and immunity and are potential drug targets. However, it has been challenging to obtain lead compounds that are sufficiently selective for a particular caspase given structural conservation in the catalytic pocket among all caspases. Thus, the GSDMD-binding hydrophobic pocket in caspase-1/4/11, which is outside of the catalytic site, suggests a new space for developing caspase drugs to treat pyroptosis-caused inflammatory diseases such as sepsis.

Graduate student Kun Wang of PTN program and Dr. Qi Sun of Shao lab are the co-first authors of this paper. Other contributors include graduate students Xiu Zhong, Mengxue Zeng, Huan Zeng, Xuyan Shi, Dr. Zilin Li, and Dr. Yupeng Wang from Shao lab. Dr. Qiang Zhao from Organ Transplant Center of The First Affiliated Hospital of Sun Yat-sen University provides the healthy and pathological liver samples. Dr. Feng Shao and visiting scientist Dr. Jingjin Ding from Institute of Biophysics (CAS) are the co-corresponding authors. The study was supported by the Strategic Priority Research Program of CAS, Basic Science Center Project and Excellent Young Scholar Program of NSFC, the National Key Research and Development Programs of China, the program Initiative for Innovative Medicine of Chinese Academy of Medical Sciences, the program Youth Innovation Promotion Association of CAS, and carried out at National Institute of Biological Sciences, Beijing.