Gasdermins: Effectors of Pyroptosis.

The terminal differentiation of neuronal and pancreatic beta-cells requires the specific expression of genes that are targets of an important transcriptional repressor named RE-1 silencing transcription factor (REST). The molecular mechanism by which these REST target genes are expressed only in neuronal and beta-cells and are repressed by REST in other tissues is a central issue in differentiation program of neuronal and beta-cells. Herein, we showed that the transcriptional factor Sp1 was required for expression of most REST target genes both in insulin-secreting cells and neuronal-like cells where REST is absent. Inhibition of REST in a non-beta and a non-neuronal cell model restored the transcriptional activity of Sp1. This activity was also restored by trichostatin A indicating the requirement of histone deacetylases for the REST-mediated silencing of Sp1. Conversely, exogenous introduction of REST blocked Sp1-mediated transcriptional activity. The REST inhibitory effect was mediated through its C-terminal repressor domain, which could interact with Sp1. Taken together, these data show that the inhibition of Sp1 by REST is required for the silencing of its target genes expression in non-neuronal and in non-beta-cells. We conclude that the interplay between REST and Sp1 determines the cell-specific expression of REST target genes.

Gasdermins (GSDMs) are a group of proteins that are cleaved by inflammatory caspases to induce pore formation in the plasma membrane to cause membrane permeabilization and lytic cell death or pyroptosis. All GSDMs share a conserved structure, containing a cytotoxic N-terminal (NT) pore-forming domain and a C-terminal (CT) repressor domain. Entamoeba histolytica (Eh) in contact with macrophages, triggers outside-in signaling to activate inflammatory caspase-4/1 via the noncanonical and canonical pathway to promote cleavage of gasdermin D (GSDMD). Cleavage of GSDMD removes the auto-inhibition that masks the active pore-forming NT domain in the full-length protein by interactions with GSDM-CT. The cleaved NT-GSDMD monomers then oligomerize to form pores in the plasma membrane to facilitate the release of IL-1β and IL-18 with a measured amount of pyroptosis. Pyroptosis is an effective way to counteract intracellular parasites, which exploit replicative niche to avoid killing. To date, most GSDMs have been verified to perform pore-forming activity and GSDMD-induced pyroptosis is rapidly emerging as a mechanism of anti-microbial host defence. Here, we review our comprehensive and current knowledge on the expression, activation, biological functions, and regulation of GSDMD cleavage with emphases on physiological scenario and related dysfunctions of each GSDM member as executioner of cell death, cytokine secretion and inflammation against Eh and other protozoan parasitic infections.

The gasdermin family (GSDMs) contains a group of uncharacterized proteins that form membrane pores and serve as a major substrate for inflammatory caspases and the execution of pyroptosis, which is recognized as a novel type of programmed cell death [1]. Gasdermin (GSDM) family consists of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C GSDMC), Gasdermin D (GSDMD), Gasdermin E(GSDME) and Pejvakin (PJVK). With the exception of PJVK, all GSDMs consist of two conserved domains: the C-terminal inhibitory domain (RD) and the N-terminal effector domain (PFD), where the N-terminal domain is cytotoxic, while the full-length structure It is not cytotoxic, indicating that the C-terminus of the GSDMs protein family (GSDMs-C) has auto-inhibitory and protective effects [2]. Because of the presence of the C-terminus, the GSDM protein does not cause cell death if it is not cleaved. Once RD is removed by hydrolysis, its PFD can combine with lipid components to form pores in the cell membrane [3]. Current studies have found that, except for PJVK, the N-terminal domains of almost all GSDMs have the ability to form pores in the plasma membrane. Among GSDMs, only the mechanism of GSDMD-induced pyroptosis is relatively clear.

Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.

Pathogen recognition receptors are important first responders to viral infection that bind to viral proteins or nucleic acids to trigger the immune response. RIG-1 is an RNA helicase with tandem caspase activation and recruitment domains (CARDs) that binds viral RNA and ultimately stimulates the genes induced by interferon regulator factor 3 (IRF-3) and nuclear factor κB (NF-κB). The CARDs of RIG-1 interact with the CARD of interferon (IFN)-β promoter stimulator 1 (IPS-1), an adaptor that links RIG-1 to the signaling pathway that leads to gene activation. Saito et al . report that RIG-1 is maintained inactive as a result of the interaction of a C-terminal repressor domain (RD), and this autoinhibition is alleviated by binding to RNA with a secondary structure, including RNA from hepatitis C virus. A related helicase, LGP2, which lacks CARDs, also bound double-stranded RNA, including sequences from hepatitis C virus. Mutation analysis showed that RNA binding by RIG-1 required the helicase domain and the C terminus but did not require the CARDs. Trypsin digestion studies indicated that upon binding RNA, RIG-1 underwent a conformational change that displaced the C-terminal region. Downstream signaling, the interaction with IPS-1, and gene activation by RIG-1 in response to Sendai virus infection required both CARDs. However, a RIG-1 with a C-terminal deletion was constitutively activated, and the C-terminal domain served as a dominant-negative inhibiting RIG-1. In transfected cells, Sendai virus induced the formation of RIG-1 multimers, and this was blocked by coexpression of the C-terminal RD. The C-terminal domain of LGP2 is similar to that of RIG-1, and when transfected into cells expressing RIG-1, LGP2 prevented induction of gene expression in response to viral infection. Full-length LGP2 or the C-terminal RD region formed a complex with RIG-1 when the proteins were overexpressed. Expression of the RIG-1 RD in cells conferred increased permissiveness to viral infection with Sendai virus. Thus, the RD of RIG-1 keeps RIG-1 inactive until viral RNA is present; then a conformational change alleviates this repression, allowing RIG-1 to interact with IPS-1 and stimulate genes involved in the immune response. LGP2, through its RD, appears to act in trans to inhibit RIG-1. Thus, these two proteins appear to serve as modulators of the response--RIG-1 turns it on, and LGP2 turns it off. T. Saito, R. Hirai, Y.-M. Loo, D. Owen, C. L. Johnson, S. C. Sinha, S. Akira, T. Fujita, M. Gale Jr., Regulation of innate antiviral defenses through a shared repressor domain in RIG-1 and LGP2. Proc. Natl. Acad. Sci. U.S.A. 104 , 582-587 (2007). [Abstract] [Full Text]

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

RIG‐I (Retinoic acid Inducible Gene‐I) is a cytoplasmic pathogen recognition receptor that differentiates between viral and cellular RNAs to trigger the innate immune response. RIG‐I comprises N‐terminal CAspase Recruitment Domains (CARDs), a DExH/D box helicase/ATPase domain and a C‐terminal Repressor Domain (RD). The helicase and repressor domains (RD) of RIG‐I recognize double‐stranded (ds) RNA and 5′‐triphosphate (ppp) RNA as foreign and activate the RIG‐I CARDs domain for downstream signaling. To understand the synergy between helicase and RD for RNA binding and how ATP hydrolysis contributes to RIG‐I activation, we determined the crystal structure of human RIG‐I helicase‐RD in complex with dsRNA and an ATP‐analog. Helicase‐RD organizes into a ring around dsRNA that caps the blunt‐end with the helicase utilizing previously uncharacterized motifs to recognize dsRNA. Small angle X‐ray scattering (SAXS), limited proteolysis, and differential scanning fluorimetry suggest that RIG‐I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full‐length RIG‐I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG‐I helicase‐RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.

The zinc finger protein REST (RE-1 silencing transcription factor) is a transcriptional repressor that inhibits neuronal gene transcription in non-neuronal tissues. REST may represent a master regulator of neuronal gene expression. REST contains two repressor domains located at the N- and C-termini of the molecule. To investigate the molecular mechanism of transcriptional repression by REST, in vivo competition experiments were performed. Both repression domains were expressed in the nucleus as fusion proteins with S. japonicum glutathione S-transferase (GST). The ability of these fusion proteins to block transcriptional repression mediated by the repressor domains of REST was tested. The results show that transcriptional repression by the N-terminal repression domain of REST could be overcome by expression of a GST fusion protein encoding the N-terminal, but not C-terminal repression domain, and vice versa, suggesting that both repression domains have to interact with distinct nuclear factors to exhibit biological activity. The GST-REST fusion proteins had no effect upon transcriptional repression mediated by the KRAB (Krüppel-associated box) domain, a strong mammalian repressor domain, or the repressor domain derived from the thyroid hormone receptors alpha. We conclude that REST has to interact with at least two distinct nuclear factors to inhibit transcription. These factors are distinct from the mammalian corepressor proteins KAP-1/KRIP-1 and N-CoR that mediate repression by the KRAB domain or the thyroid hormone receptor alpha. Thus, mammalian transcriptional repressors utilize different mechanisms to inhibit transcription by using different kinds of protein-protein interactions.

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2.

Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ERalpha)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERalpha activity. SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ERalpha activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Structural Basis Of RIG-I Auto-inhibition And RNA-induced Activation

The gasdermins are a family of pore-forming proteins that have recently emerged as executors of pyroptosis, a lytic form of cell death that is induced by the innate immune system to eradicate infected or malignant cells. Mammalian gasdermins comprise a cytotoxic N-terminal domain, a flexible linker and a C-terminal repressor domain. Proteolytic cleavage in the linker releases the cytotoxic domain, thereby allowing it to form β-barrel membrane pores. Formation of gasdermin pores in the plasma membrane eventually leads to a loss of the electrochemical gradient, cell death and membrane rupture. Here we review recent work that has expanded our understanding of gasdermin biology and function in mammals by revealing their activation mechanism, their regulation and their roles in autoimmunity, host defence and cancer. We further highlight fungal and bacterial gasdermin pore formation pointing to a conserved mechanism of cell death induction.

Li, G., Li, J., Tian, Y., Wang, DE., Shen, J., Tao, Z., Xu, J. and Lu, L. 2012. Sequence analysis of a putative goose RIG-I gene. Can. J. Anim. Sci. 92: 143–151. Retinoid acid-inducible gene-I (RIG-I) is a critical cytoplasmic RNA sensor which plays an important role in the recognition of, and response to, influenza virus and other RNA viruses. In the present study, A 3808-bp cDNA encoding goose RIG-I (goRIG-I) was cloned from splenic lymphocytes of geese using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The encoded protein, which is predicted to consist of 933 amino acids, has a molecular weight of 106.4 kDa and includes an N-terminal caspase recruitment domain (CARD), a domain with the signature of DExD/H box helicase (helicase domain), and a C-terminal repression domain (RD) similar to duck RIG-I (duRIG-I), human RIG-I, and mouse RIG-I. The goRIG-I showed 93.8 and 78.0% amino acid sequence identity with previously described duRIG-I and finch RIG-I, respectively, and 48.9–53.0% sequence identity with mammalian homologs. Quantitative RT-PCR analysis indicated that the goRIG-I gene is strongly expressed in the liver, lung, brain, spleen, and bursa of Fabricius. These findings lay the foundation for further research on the function and mechanism of avian RIG-I in innate immunity.

The Kruppel-associated box (KRAB)-zinc finger protein ZBRK1 has been implicated in the transcriptional regulation of DNA damage-response genes that function in cell growth control and survival. Recently, we described a novel BRCA1-dependent C-terminal transcriptional repression domain (CTRD) within ZBRK1, the mode of repression of which is functionally distinguishable from that of the N-terminal KRAB repression domain within ZBRK1. The identification of BRCA1 binding-competent but repression-defective CTRD mutants further revealed that BRCA1 binding is necessary, but not sufficient, for ZBRK1 CTRD function. During an unbiased search for possible co-regulators of the CTRD, we identified ZBRK1 itself, suggesting that ZBRK1 can oligomerize through its CTRD. Herein we explore the physical and functional requirements for ZBRK1 oligomerization in ZBRK1-directed transcriptional repression. Protein interaction analyses confirmed that ZBRK1 can homo-oligomerize both in vitro and in vivo and further mapped the ZBRK1 oligomerization domain to the CTRD C terminus. Biochemical analyses, including protein cross-linking and gel filtration chromatography, revealed that ZBRK1 homo-oligomers exist as tetramers in solution. Functionally, ZBRK1 oligomerization facilitates ZBRK1-directed transcriptional repression through ZBRK1 response elements; requirements for oligomerization-dependent repression include the ZBRK1 CTRD and KRAB repression domains but not the DNA binding activity of ZBRK1. These observations suggest that higher order oligomers of ZBRK1 may assemble on target ZBRK1 response elements through both protein-DNA and CTRD-dependent protein-protein interactions. These findings thus reveal an unanticipated dual function for ZBRK1 in both DNA binding-dependent and -independent modes of transcriptional repression and further establish the CTRD as a novel protein interaction surface responsible for directing homotypic and heterotypic interactions necessary for ZBRK1-directed transcriptional repression.