Gasdermin and Gasdermin-Like Pore-Forming Proteins in Invertebrates,Fungi and Bacteria

The gasdermin family of pore-forming proteins (PFPs) has recently emerged as key molecular players controlling immune-related cell death in mammals. Characterized mammalian gasdermins are activated through proteolytic cleavage by caspases or serine proteases, which remove an inhibitory carboxy-terminal domain, allowing the pore-formation process. Processed gasdermins form transmembrane pores permeabilizing the plasma membrane, which often results in lytic and inflammatory cell death. While the gasdermin-dependent cell death (pyroptosis) has been predominantly characterized in mammals, it now has become clear that gasdermins also control cell death in early vertebrates (teleost fish) and invertebrate animals such as corals (Cnidaria). Moreover, gasdermins and gasdermin-like proteins have been identified and characterized in taxa outside of animals, notably Fungi and Bacteria. Fungal and bacterial gasdermins share many features with mammalian gasdermins including their mode of activation through proteolysis. It has been shown that in some cases the proteolytic activation is executed by evolutionarily related proteases acting downstream of proteins resembling immune receptors controlling pyroptosis in mammals. Overall, these findings establish gasdermins and gasdermin-regulated cell death as an extremely ancient mechanism of cellular suicide and build towards an understanding of the evolution of regulated cell death in the context of immunology. Here, we review the broader gasdermin family, focusing on recent discoveries in invertebrates, fungi and bacteria.     

Mammalian initiator apoptotic caspases

Caspases are a conserved family of cysteine proteases. They play diverse roles in inflammatory responses and apoptotic pathways. Among the caspases is a subgroup whose primary function is to initiate apoptosis. Within their long prodomains, caspases-2, -9 and -12 contain a caspase activation and recruitment domain while caspases-8 and -10 bear death effector domains. Activation follows the recruitment of the procaspase molecule via the prodomain to a high molecular mass complex. Despite sharing some common features, other aspects of the biochemistry, substrate specificity, regulation and signaling mechanisms differ between initiator apoptotic caspases. Defects in expression or activity of these caspases are related to certain pathological conditions including neurodegenerative disorders, autoimmune diseases and cancer.     

Epithelial Pyroptosis in Host Defense

Pyroptosis is a lytic form of cell death that is executed by a family of pore-forming proteins called gasdermins (GSDMs). GSDMs are activated upon proteolysis by host proteases including the proinflammatory caspases downstream of inflammasome activation. In myeloid cells, GSDM pore formation serves two primary functions in host defense: the selective release of processed cytokines to initiate inflammatory responses, and cell death, which eliminates a replicative niche of the pathogen. Barrier epithelia also undergo pyroptosis. However, unique mechanisms are required for the removal of pyroptotic epithelial cells to maintain epithelial barrier integrity. In the following review, we discuss the role of epithelial inflammasomes and pyroptosis in host defense against pathogens. We use the well-established role of inflammasomes in intestinal epithelia to highlight principles of epithelial pyroptosis in host defense of barrier tissues, and discuss how these principles might be shared or distinctive across other epithelial sites.     

Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.     

Regulation of Lytic and Non-Lytic Functions of Gasdermin Pores

Pyroptosis is a necrotic form of cell death that was initially found to be induced upon activation of inflammatory caspases by inflammasome complexes. Mechanistically, pyroptosis induction requires cleavage of the caspase substrate gasdermin D (GSDMD), and the release of the GSDMD N-terminal fragment, which targets the plasma membrane to form large β-barrel pores. GSDMD shares this pore-forming ability with other gasdermin family members, which induce pyroptosis during infection or upon treatment with chemotherapy drugs. While induction of cell death has been assumed to be the main function of the gasdermin pores, increasing evidence suggests that these pores have non-lytic functions, such as in releasing cytokines or alarmins and in regulating intracellular signaling via ionic fluxes. Here we discuss how gasdermin pore formation is regulated to induce membrane permeabilization or lysis, how gasdermin pores achieve specificity for cargo-release and how cells repair gasdermin-induced damage to the plasma membrane.     

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Caspases - controlling intracellular signals by protease zymogen activation

Animal development and homeostasis is a balance between cell proliferation and cell death. Physiologic, and sometimes pathologic, cell death - apoptosis - is driven by activation of a family of proteases known as the caspases, present in almost all nucleated animal cells. The enzymatic properties of these proteases are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side chain for catalyzing peptide bond cleavage. The primary specificity for Asp turns out to be very rare among proteases, and currently the only other known mammalian proteases with the same primary specificity is the physiological caspase activator granzyme B. Like most other proteases, the caspases are synthesized as inactive zymogens whose activation requires limited proteolysis or binding to co-factors. To transmit the apoptotic execution signal, caspase zymogens are sequentially activated through either an intrinsic or an extrinsic pathway. The activation of caspases at the apex of each pathway, the initiators, occurs by recruitment to specific adapter molecules through homophilic interaction domains, and the activated initiators directly process the executioner caspases to their catalytically active forms. In the present communication we review the different mechanisms underlying the selective activation of the caspases.     

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains,modules, and exosites

The function of ancillary domains and modules attached to the catalytic domain of mutidomain proteases, such as the matrix metalloproteinases (MMPs), are not well understood. The importance of discrete MMP substrate binding sites termed exosites on domains located outside the catalytic domain was first demonstrated for native collagenolysis. The essential role of hemopexin carboxyl-domain exosites in the cleavage of noncollagenous substrates such as chemokines has also been recently revealed. This article updates a previous review of the role of substrate recognition by MMP exosites in both preparing complex substrates, such as collagen, for cleavage and for tethering noncollagenous substrates to MMPs for more efficient proteolysis. Exosite domain interaction and movements—“molecular tectonics”—that are required for native collagen triple helicase activity are discussed. The potential role of collagen binding in regulating MMP-2 (gelatinase A) activation at the cell surface reveals unexpected consequences of substrate interactions that can lead to collagen cleavage and regulation of the activation and activity of downstream proteinases necessary to complete the collagenolytic cascade.     

Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase subgroups. To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups. The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse residues. Glu is not required at the P3 position because Ac-DMQD-Cho, Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7 has alternate regions for binding of small branched aliphatic or polar residues similar to those of initiator caspase-8. The observed plasticity of the caspase subsites agrees very well with the reported cleavage of many proteins at noncanonical sites. The results imply that factors other than the P4-P1 sequence, such as exosites, contribute to the in vivo substrate specificity of caspases. The novel peptide binding site identified on the molecular surface of the current structures is suggested to be an exosite of caspase-7. These results should be considered in the design of selective small molecule inhibitors of this pharmacologically important protease.     

Plant phytaspases and animal caspases: structurally unrelated death proteases with a common role and specificity

Proteases with an aspartate cleavage specificity are known to contribute to programmed cell death (PCD) in animals and plants. In animal cells this proteolytic activity belongs to caspases, a well-characterized family of cysteine-dependent death proteases. Plants, however, lack caspase homologs and thus the origin of this type of proteolytic activity in planta was poorly understood. Here, we review recent data demonstrating that a plant serine-dependent protease, phytaspase, shares cleavage specificity and a role in PCD analogous to that of caspases. However, unlike caspases, regulation of phytaspase-mediated cleavage of intracellular target proteins appears to be attained not at the level of proenzyme processing/activation, which occurs, in the case of phytaspase, autocatalytically and constitutively. Rather, the mature phytaspase is excluded from healthy cells into the apoplast and is allowed to re-enter cells upon the induction of PCD. Thus, PCD-related proteases in animals and plants display both common features and important distinctions.     

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.     

The inflammatory caspases: key players in the host response to pathogenic invasion and sepsis

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). The inflammatory caspases were among the first to be discovered, but only recently have the mechanisms leading to their activation and inhibition begun to be elucidated. In this review, we examine the biochemistry, substrates, and function of this unique family of inflammatory proteases, highlight the most recent findings regarding their regulatory mechanisms, and discuss what remains to be understood about their roles in health and disease.     

Pyroptosis in Steatohepatitis and Liver Diseases

Pyroptosis is an inflammatory form of regulated cell death, which functions in the clearance of intracellularly replicating pathogens by cell lysis in order to induce further immune response. Since the discovery of the gasdermin (GSDM) family, pyroptosis has attracted attention in a wide range of inflammatory diseases such as nonalcoholic steatohepatitis and other liver diseases. Due to the cleavage of GSDMs by different caspases, the amino-terminal GSDM fragments form membrane pores essential for pyroptosis that facilitate the release of inflammatory cytokines by loss of ionic gradient and membrane rupture. In this review, we address the key molecular and cellular processes that induce pyroptosis in the liver and its significance in the pathogenesis of common liver diseases in different human and experimental mice studies.     

The CASBAH: a searchable database of caspase substrates

Apoptosis is coordinated by members of the caspase family of aspartic acid-specific proteases. Other members of this protease family also play essential roles in inflammation where they participate in the maturation of pro-inflammatory cytokines. To date, almost 400 substrates for the apoptosis-associated caspases have been reported and there are likely to be hundreds more yet to be discovered. Thus, the fraction of the proteome that is degraded (the degradome) by caspases during the demolition phase of apoptosis appears to be quite substantial. Despite this, we still know surprisingly little concerning how caspases provoke some of the signature events in apoptosis, such as membrane phosphatidylserine externalization, cellular retraction, chromatin condensation and apoptotic body production. The inflammatory caspases appear to be much more specific proteases than those involved in apoptosis and only two confirmed substrates for these proteases have been described to date. Here, we have compiled a comprehensive list of caspase substrates and describe a searchable web resource (The Casbah; www.casbah.ie) which contains information pertaining to all currently known caspase substrates. We also discuss some of the unresolved issues relating to caspase-dependent events in apoptosis and inflammation.     

Activity of ADAM17 (a Disintegrin and Metalloprotease 17) Is Regulated by Its Noncatalytic Domains and Secondary Structure of its Substrates

ADAM proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off target toxicity. Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strategy for drug discovery; however, there are only a few reports of potential exosites in ADAM protease structures. In the study presented here, we utilized a series of TNFα-based substrates to probe ADAM10 and 17 interactions with its canonical substrate to identify the structural features that determine ADAM protease substrate specificity. We found that noncatalytic domains of ADAM17 did not directly bind the substrates used in the study but affected the binding nevertheless, most likely because of steric hindrance. Additionally, noncatalytic domains of ADAM17 affected the size/shape of the carbohydrate-binding pocket contained within the catalytic domain of ADAM17. This suggests that noncatalytic domains of ADAM17 play a role in substrate specificity and might help explain differences in substrate repertoires of ADAM17 and its closest homologue, ADAM10. We also addressed the question of which substrate features can affect ADAM protease specificity. We found that all ADAM proteases tested (i.e., ADAM10, 12, and 17) significantly decreased activity when the TNFα-derived sequence was induced into α-helical conformation, suggesting that conformation plays a role in determining ADAM protease substrate specificity. These findings can help in the discovery of ADAM isoform- and substrate-specific inhibitors.     

Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.