Cellular pyroptosis is a programmed cell death mediated by the Gasdermin (GSDM) family of proteins, which plays a role in the body's defense against pathogenic infections, clearance of mutated or harmful cells, and other processes. As the direct executor of cell pyroptosis, GSDM protein has received much attention. The GSDM protein in mammals has a conserved self inhibitory dual domain feature, and the inhibitory C-terminal domain locks the full-length protein in a non activated state through intramolecular interactions with the N-terminal effector domain. The activation of GSDM protein requires upstream specific protease cleavage, release of N-terminal effector domains, and oligomerization drilling on the cell membrane, mediating cell pyroptosis. GSDM is a class of evolutionarily conserved membrane drilling proteins that exist in various bacteria, fungi, invertebrates, and all vertebrates. Although the sequence differences between GSDM proteins found in lower organisms such as bacteria and fungi and GSDM proteins in mammals are significant, and their C-terminus structural elements are generally short, they are maintained in a typical self inhibition manner in a non activated state, requiring specific protease cleavage to release the conserved membrane pore domains, causing pyroptosis like lytic cell death. Therefore, further research is needed to determine whether there are other activation mechanisms of GSDM protein that do not depend on protease cleavage, and how this mechanism mediates the function of GSDM protein in performing cell pyroptosis, in addition to protease cleavage activation.

On April 25, Ding Jing, the research team of the Institute of Biophysics of the Chinese Academy of Sciences, in cooperation with Shao Feng's team of the Beijing Institute of Life Sciences, published a collaborative research paper entitled "Clearance independent activation of artificial eucaryotic gasterminals and structural mechanisms" online in Science, revealing the molecular mechanism of two GSDM proteins from lower eukaryotes activated by novel ways of non protease cleavage.

This study found through sequence homology analysis that the genome of the most primitive multicellular organism, TrichoGSDM, encodes a GSDM homologous protein that only contains a membrane perforated domain (TrichoGSDM). The study found through recombinant expression and purification identification that the TrichoGSDM protein exists in both monomer and dimer forms. Among them, monomeric proteins have the activity of drilling holes on liposomes, while dimers cannot drill holes on the membrane. Furthermore, the high-resolution crystal structure of TrichoGSDM dimer was studied and analyzed, and it was found that TrichoGSDM dimer is formed by crosslinking two monomer proteins through three pairs of intermolecular disulfide bonds. Research has shown that using reducing agents to treat TrichoGSDM dimers or mutant Cys involved in disulfide bond formation in vitro can yield homogeneous monomer proteins and exhibit strong membrane punching activity. This indicates that the dimers linked by disulfide bonds represent the inactive state of TrichoGSDM proteins, and the monomer transition from dimers to reducing states may be a potential activation mechanism for TrichoGSDM proteins. Glutathione (GSH) and thioredoxin (TRX) in the cytoplasm are two antioxidant systems that can eliminate harmful reactive oxygen species or disulfide bonds formed by protein oxidation in the cytoplasm, and maintain a reducing environment in the cytoplasm. Furthermore, this study utilized the physiological concentration of GSH in the cytoplasm or the TRX protein of TrichoGSDM to treat the dimer. The dimer was reduced and the membrane perforation activity of monomers was released, which induced the expression of TrichoGSDM in bacteria with antibacterial activity similar to the N-terminal domain of mammalian GSDM protein. This indicates that the reducing environment of bacterial cytoplasm is conducive to maintaining TrichoGSDM in an activated monomer state, and inhibiting bacterial growth by perforation on the bacterial membrane. Furthermore, the high-resolution cryo electron microscopy structure of the molecular pores formed by TrichoGSDM on liposome membranes was studied and analyzed, and it was found that TrichoGSDM formed the largest known GSDM pore in eukaryotes from 44 monomers. The study revealed the structural basis for TrichoGSDM to recognize acidic phospholipids, undergo conformational changes, and oligomeric assemble into pores through structural analysis. The above study elucidates the molecular mechanism of TrichoGSDM mediated dimeric self inhibition through intermolecular disulfide bonds, activation into monomeric states with pore forming activity through reduction of disulfide bonds, and further oligomerization pore forming on the membrane to mediate cell death. This novel activation mechanism was first discovered in the GSDM protein.

The discovery of TrichoGSDM has sparked the interest of researchers to continue exploring GSDM proteins that only contain membrane perforated domains. The fusion lethal gene rcd-1 was discovered in the filamentous fungus Pseudomonas aeruginosa, and the alleles in different strains can encode two homologous proteins, RCD-1-1 and RCD-1-2. When different strains undergo cell fusion, two RCD-1 proteins mediate allogeneic recognition leading to cell death. Through analyzing the crystal structures of RCD-1-1 and RCD-1-2, it was found that the two RCD-1 proteins have similar structural features to the membrane pore domains of mammalian GSDM, but they lack structural elements that exert self inhibitory functions. The individual RCD-1-1 or RCD-1-2 exhibit a monomeric state in solution, which recognizes the binding of acidic phospholipids on the liposome membrane but cannot oligomeric punch holes, thus exhibiting no cytotoxicity. However, when two RCD-1 proteins are co expressed in various cell systems such as Escherichia coli, brewing yeast, or HeLa cells, they can cause strong lytic cell death. The study analyzed the three-dimensional structure of molecular pores formed by co incubated RCD-1-1 and RCD-1-2 proteins on liposome membranes using cryo electron microscopy. It was found that the two proteins formed the smallest known GSDM pores through alternating heterooligomeric assembly. Research and analysis of the interaction modes of two proteins in the pores of RCD-1 molecule revealed that each RCD-1-1 molecule interacts with adjacent RCD-1-2 molecules on both sides, but the interaction modes on both sides are not equivalent. That is to say, the side with stronger intermolecular polarity dominates the formation of RCD-1 heterodimers, while the intermolecular interactions on the other side drive further oligomerization into pores using heterodimers as units. The co incubation of RCD-1-1 and RCD-1-2 proteins with liposomes, or their respective binding to liposomes before co incubation, can activate the punching activity on the liposome membrane through intermolecular recognition of the two proteins. However, the key residue mutation at the heterodimer recognition interface can block the intermolecular recognition of RCD-1 protein when fusing yeast cells expressing different mating types of the two proteins or spore fusion of different rough spore strains, which cannot activate membrane punching activity and cause lytic cell death. The above work reveals that the RCD-1 protein with membrane binding properties is in an inactive resting state when it exists alone. Cell fusion leads to the meeting of two proteins, which activate heterodimer assembly through molecular specific recognition and further oligomerize into pores on the cell membrane, performing the function of cell death.

The above achievements break the traditional understanding that GSDM proteins require protease cleavage to open self inhibition and activate membrane pore activity. They reveal two types of GSDM proteins in lower eukaryotes that only contain membrane pore domains, which release membrane pore activity through redox regulation or paired molecular interactions, respectively. This new activation mechanism expands our understanding of the evolution and functional diversity of GSDM proteins. Multiple activation mechanisms indicate that GSDM proteins can respond to a wider range of biological signals and participate in richer life processes. Meanwhile, this enzyme independent GSDM protein has the potential to be developed as a novel tool for inducing cell death, and is expected to contribute to basic research and translational studies related to cell pyroptosis.



The research work was supported by the National Natural Science Foundation of China, the Ministry of Science and Technology, the Chinese Academy of Sciences and the Chinese Academy of Medical Sciences.



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