Caspases in Cell Death, Inflammation, and Pyroptosis.

Caspases are a family of conserved cysteine proteases that play key roles in programmed cell death and inflammation. In multicellular organisms, caspases are activated via macromolecular signaling complexes that bring inactive procaspases together and promote their proximity-induced autoactivation and proteolytic processing. Activation of caspases ultimately results in programmed execution of cell death, and the nature of this cell death is determined by the specific caspases involved. Pioneering new research has unraveled distinct roles and cross talk of caspases in the regulation of programmed cell death, inflammation, and innate immune responses. In-depth understanding of these mechanisms is essential to foster the development of precise therapeutic targets to treat autoinflammatory disorders, infectious diseases, and cancer. This review focuses on mechanisms governing caspase activation and programmed cell death with special emphasis on the recent progress in caspase cross talk and caspase-driven gasdermin D-induced pyroptosis.

Molecular mechanism of bovine Gasdermin D-mediated pyroptosis.

Pyroptosis is a form of programmed cell death characterized by cell swelling, pore formation in the plasma membrane, lysis, and releases of cytoplasmic contents. To date, the molecular mechanism of human and murine Gasdermin D-mediated pyroptosis have been fully investigated. However, studies focusing on molecular mechanism of bovine Gasdermin D (bGSDMD)-mediated pyroptosis and its function against pathogenic infection were unclear. In the present study, we demonstrate that bovine caspase-1 (bCaspase-1) cleaves bGSDMD at amino acid residue D277 to produce an N-terminal fragment (bGSDMD-p30) which leads to pyroptosis. The amino acid residues T238 and F239 are critical for bGSDMD-p30-mediated pyroptosis. The loop aa 278-299, L293 and A380 are the key sites for autoinhibitory structure of the full length of bGSDMD. In addition, bCaspase-3 also cleaves bGSDMD at residue Asp86 without inducing cell death. Therefore, our study provides the first detailed elucidation of the mechanism of bovine GSDMD-mediated pyroptosis. The results will establish a significant foundation for future research on the role of pyroptosis in bovine infectious diseases.

Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function.

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1ß and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.

Molecular Characteristics of Cell Pyroptosis and Its Inhibitors: A Review of Activation, Regulation, and Inhibitors.

Pyroptosis is an active and ordered form of programmed cell death. The signaling pathways of pyroptosis are mainly divided into canonical pathways mediated by caspase-1 and noncanonical pathways mediated by caspase-11. Cell pyroptosis is characterized by the activation of inflammatory caspases (mainly caspase-1, 4, 5, 11) and cleavage of various members of the Gasdermin family to form membrane perforation components, leading to cell membrane rupture, inflammatory mediators release, and cell death. Moderate pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. Based on canonical pathways, research on pyroptosis regulation has demonstrated several pyroptotic inhibitors, including small-molecule drugs, natural products, and formulations of traditional Chinese medicines. In this paper, we review the characteristics and molecular mechanisms of pyroptosis, summarize inhibitors of pyroptosis, and propound that herbal medicines should be a focus on the research and development for pyroptosis blockers.

Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.

Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation.

Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma.

Dual function of a turbot inflammatory caspase in mediating both canonical and non-canonical inflammasome activation.

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1ß release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity.

Regulatory roles of ginseng on inflammatory caspases, executioners of inflammasome activation.

Inflammation is an immune response that protects against pathogens and cellular stress. The hallmark of inflammatory responses is inflammasome activation in response to various stimuli. This subsequently activates downstream effectors, that is, inflammatory caspases such as caspase-1, 4, 5, 11, and 12. Extensive efforts have been made on developing effective and safe anti-inflammatory therapeutics, and ginseng has long been traditionally used as efficacious and safe herbal medicine in treating various inflammatory and inflammation-mediated diseases. Many studies have successfully shown that ginseng plays an anti-inflammatory role by inhibiting inflammasomes and inflammasome-activated inflammatory caspases. This review discusses the regulatory roles of ginseng on inflammatory caspases in inflammatory responses and also suggests new research areas on the anti-inflammatory function of ginseng, which provides a novel insight into the development of ginseng as an effective and safe anti-inflammatory herbal medicine.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including ep-ithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-termi-nal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pio-neering a new area for structural and functional research on Gasdermin family proteins. This review will sum-marize the latest progress in the structures, functions and association with diseases of several Gasdermin fam-ily proteins.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including epithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-terminal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pioneering a new area for structural and functional research on Gasdermin family proteins. This review will summarize the latest progress in the structures, functions and association with diseases of several Gasdermin family proteins.

Gasdermin D:The killer protein performs cell pyroptosis / 医学研究生学报

Pyroptosis is a novel programmed cell death mode mediated by inflammatory Caspase,which has a proinflammatory effect and is accompanied by cell membrane rupture and disintegration.In recent years,researchers found that the substrate of inflam-matory Caspase-Gasdermin D(GSDMD)protein,is the key"killer protein"to execute cell pyroptosis.The inflammatory Caspase spe-cifically cleaves the GSDMD protein,forming the N-terminal domain and the C-terminal domain,and eventually results in cell pyropto-sis by protein pores formation.This article focuses on the research progress of cell pyroptosis,the structural features of GSDMD protein, and the mechanism of GSDMD protein participating in the pyroptosis and the disease models.