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Various proteins are involved in fish venom toxicity, but limited information is available regarding their structure and mode of action. Here, we analyzed RNA transcripts in the dorsal spine of the devil stinger Inimicus japonicus using next-generation sequencing (NGS), and identified two putative protein toxins, a natterin-like protein (Ij-natterin) and a phospholipase A2 (Ij-PLA2), as well as a previously reported stonustoxin-like protein. The deduced amino acid sequence of Ij-natterin suggested that it acts as a pore-forming toxin through the cooperation of the N-terminal lectin-like domain and the C-terminal pore-forming domain. Ij-PLA2 showed significant homology with secreted Ca2+-dependent PLA2s from snake venom and mammals (sPLA2-I/II). The recombinant Ij-PLA2 protein exhibited PLA2 activity in the absence of Ca2+, in contrast to canonical sPLA2-I/II. Comparison of the amino acid sequences of Ij-PLA2 with the other sPLA2-I/II suggests that the C-terminal extended peptide region of Ij-PLA2 is involved in its Ca2+-independent activity.

The integrity of the plasma membranes is extremely crucial for the survival and proper functioning of the cells. Organisms from all kingdoms of life employ specialized pore-forming proteins and toxins (PFPs and PFTs) that perforate cell membranes, and cause detrimental effects. PFPs/PFTs exert their damaging actions by forming oligomeric pores in the membrane lipid bilayer. PFPs/PFTs play important roles in diverse biological processes. Many pathogenic bacteria secrete PFTs for executing their virulence mechanisms. The immune system of the higher vertebrates employs PFPs to kill pathogen-infected cells and transformed cancer cells. The most obvious consequence of membrane pore-formation by the PFPs/PFTs is the killing of the target cells due to the disruption of the permeability barrier function of the plasma membranes. PFPs/PFTs can also activate diverse cellular processes that include activation of the stress-response pathways, induction of programmed cell death, and inflammation. Upon attack by the PFTs, host cells may also activate pathways to repair the injured membranes, restore cellular homeostasis, and trigger inflammatory immune responses. In this article, we present an overview of the diverse cellular responses that are triggered by the PFPs/PFTs, and their implications in the process of pathogen infection and immunity.

Aim To test whether engineered nanopores on the outer membrane (OM) of Escherichia coli can increase expression of heterologous proteins by making additional nutrients available to the host. Methods and results Outer membrane nanopores were generated by expressing recombinant Vibrio parahaemolyticus CsgG (rVpCsgG), which spontaneously assembles into a pore-forming channel on the OM, allowing spontaneous diffusion of small chemical entities from the exterior. Protein expression was probed using a reporter protein, sfGFP, expressed on a second compatible plasmid. OM pore formation was shown by acquired erythromycin sensitivity in cells transformed with rVpCsgG, influx of propidium iodide as well as by surface localization of recombinant CsgG by immunogold-labeled transmission electron microscopy. Expression of recombinant CsgG showed increased growth and also enhanced expression of sfGFP in minimal medium and is due to both enhanced transcription as well as translation. Similar enhancement of expression was also observed for a number of different proteins of different origin, sizes and nature. Conclusions Our findings clearly demonstrate that engineered nanopores on the OM of E. coli enhance expression of different heterologous proteins in minimal medium. Significance and impact of the study Vibrio parahaemolyticus CsgG β-nanopore mediated co-expression strategy to improve recombinant protein expression is fully compatible with other methods of protein expression enhancement, and therefore can be a useful tool in biotechnology particularly for whole-cell bio-transformations for production of secondary metabolite.

Pore-forming proteins/toxins (PFPs/PFTs) are the distinct class of membrane-damaging proteins. They act by forming oligomeric pores in the plasma membranes. PFTs and PFPs from diverse organisms share a common mechanism of action, in which the designated pore-forming motifs of the membrane-bound protein molecules insert into the membrane lipid bilayer to create the water-filled pores. One common characteristic of these pore-forming motifs is that they are amphipathic in nature. In general, the hydrophobic sidechains of the pore-forming motifs face toward the hydrophobic core of the membranes, while the hydrophilic residues create the lining of the water-filled pore lumen. Interestingly, pore-forming motifs of the distinct subclass of PFPs/PFTs share very little sequence similarity with each other. Therefore, the common guiding principle that governs the sequence-to-structure paradigm in the mechanism of action of these PFPs/PFTs still remains an enigma. In this article, we discuss this notion using the examples of diverse groups of membrane-damaging PFPs/PFTs.

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

Gasdermins (GSDMs) belong to a protein superfamily that is found only in vertebrates and consists of GSDMA, GSDMB, GSDMC, GSDMD, DFNA5 (a.k.a. GSDME) and DFNB59 (a.k.a. Pejvakin (PJVK)) in humans. Except for DFNB59, all members of the GSDM superfamily contain a conserved two-domain structure (N-terminal and C-terminal domains) and share an autoinhibitory mechanism. When the N-terminal domain of these GSDMs is released, it possesses pore-forming activity that causes inflammatory death associated with the loss of cell membrane integrity and release of inflammatory mediators. It has also been found that spontaneous mutations occurring in the genes of GSDMs have been associated with the development of certain autoimmune disorders, as well as cancers. Here, we review the current knowledge of the expression profile and regulation of GSDMs and the important roles of this protein family in inflammatory cell death, tumorigenesis and other related diseases.

Supported lipid bilayers (SLBs) have emerged as powerful model systems to study various membrane-governed cellular events. Conducting polymers are excellent materials to establish electrical communication with SLBs. However, forming SLBs that are defect-free on the existing library of electronic polymer films, which have not been designed to interface lipids, remains a challenge. Moreover, the existing polymers are predominantly p-type conductors, hindering the development of devices that can be superior to current technologies. In this work, we synthesized an n-type semiconducting polymer based on a naphthalene 1,4,5,8 tetracarboxylic diimide bithiophene (NDI-T2) backbone functionalized with bio-inspired, lysine-based side chains (L2). The lysine chains, that are oriented on the surface of the film, facilitated the assembly of the zwitterionic lipid vesicles into an SLB. The n-type polymer also proved to be an ideal channel material for the state-of-the-art bioelectronic transducer, i.e., organic electrochemical transistor (OECT). We used the n-type, accumulation mode OECTs to assess the quality of the SLB as well as to monitor the activity of a pore forming protein integrated into the SLB. Our work marks the first demonstration of a bio-functionalized n-type polymer, specifically designed for interfacing the lipid membrane, alongside the high operational stability in biologically relevant electrolytes and sufficient performance in microscale transistors for biosensing applications.

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha(α)-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the β-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for β-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the β-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the β-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the β-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the β-tongue and the tendency to induce disorder in the β-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general.Author summaryBacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form bilayer spanning nanopores. Unregulated pore formation disrupts ionic balance that compromises the permeability of the cell, leading to cell death and infection. PFTs display remarkable structural plasticity that allow these proteins to interconvert between the two physiochemically distinct water soluble and membrane bound forms. However the molecular mechanism for this robust interconversion is poorly understood. For example, upon membrane binding, cytolysin A (ClyA), an α-PFT, shows one of the largest conformational changes in protein structure among the PFT family of proteins. In order to understand this transtition we characterized several point mutations in ClyA using experiments and molecular dynamics (MD) simulations to understand the role of two essential ClyA motifs (β tongue and N-terminus) implicated in the conformational changes responsible for oligomerization and conferring stability to the pore state. Our study reveals that the innate conformational flexibility of the β tongue results in a disordered intermediate state that facilitate the complete transition to the pore state. This tendency to disorder is compromised to varying degrees in ClyA mutants, correlating with the loss of lytic activity. Our results suggest that the finely tuned conformational flexibility in the membrane motifs of ClyA are critical to its function, revealing a broader paradigm that could be at play during membrane associated secondary structure transitions of proteins in general.

The secretory output of cell-cell interfaces must be tightly controlled in space and time to ensure functional efficacy. This is particularly true for the cytotoxic immune synapse (IS), the stereotyped junction formed between a cytotoxic lymphocyte and the infected or transformed target cell it aims to destroy1. Cytotoxic lymphocytes kill their targets by channeling a mixture of granzyme proteases and the pore forming protein perforin directly into the IS2, 3. The synaptic secretion of these toxic molecules constrains their deleterious effects to the target cell alone, thereby protecting innocent bystander cells in the surrounding tissue from collateral damage. Despite the importance of this process for immune specificity, the molecular and cellular mechanisms that establish secretory sites within the IS remain poorly understood. Here, we identified an essential role for integrin mechanotransduction in cytotoxic secretion using a combination of single cell biophysical measurements, ligand micropatterning, and functional assays. Upon ligand-binding, the αLβ2integrin LFA-1 functioned as a spatial cue, attracting lytic granules containing perforin and granzyme and inducing their fusion at closely adjacent sites within the synaptic membrane. LFA-1 molecules were subjected to pulling forces within these secretory domains, and genetic or pharmacological suppression of these forces abrogated cytotoxicity. We conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance both the potency and the security of their cytotoxic output.

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a secretory aerolysin family pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water intake and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporins, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

Pyroptosis is a form of programmed cell death that is highly proinflammatory. Pyroptosis was originally observed in macrophages in response to bacterial infection or bacterial toxin stimulation. This cell death, then misclassified as a type of apoptosis specifically in macrophages, requires caspase-1 that also processes interleukin-1β mainly expressed in monocytic cells. Caspase-1 is activated by canonical inflammasomes, a major cytosolic innate immune surveillance mechanism that can detect various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Studies on innate immunity to cytosolic lipopolysaccharide (LPS) have established that murine caspase-11 and its human counterparts caspase-4/5 can also mediate pyroptosis upon directly binding to the lipid A moiety of LPS. Caspase-11/4/5 do not process interleukin and caspase-11/4/5-mediated pyroptosis is prevalent in non-monocytic cells. Very recent studies have identified gasdermin D (GSDMD) as the pyroptotic substrate for both caspase-1 and caspase-11/4/5. GSDMD represents a large gasdermin family that shares membrane pore-forming activity, rendering a new concept of pyroptosis as gasdermin-mediated programmed necrotic cell death. In this article, we review the concept evolvement of pyroptotic cell death from a historic perspective, covering the innate immune pathways upstream of caspase-1 and caspase-11/4/5. We also discuss the recent advances on pyroptosis, particularly mechanistic and functional understandings of GSDMD and the gasdermin family of pyroptosis executor proteins.

Schwannomas are peripheral nerve sheath tumors associated with three genetically distinct disease entities, namely sporadic schwannoma, neurofibromatosis type-2, and schwannomatosis. Schwannomas are associated with severe disability and in cases lead to death. The primary treatment is operative resection that itself can cause neurologic damage and is at times contra-indicated due to tumor location. Given their homogenous Schwann-lineage cellular composition, schwannomas are appealing targets for gene therapy. In the present study, we have generated an adeno-associated serotype 1 virus (AAV1)-based vector delivering N-terminal of the pyroptotic gene Gasdermin-D; (GSDMDNterm) under the control of the Schwann-cell specific promoter, P0. we have demonstrated that AAV1-P0-GSDMDNterm injection into intra-sciatic schwannomas reduces the growth of these tumors and resolves tumor-associated pain without causing neurologic damage. This AAV1-P0-GSDMDNterm vector holds promise for clinical treatment of schwannomas via direct intra-tumoral injection.

Perforin, a pore-forming glycoprotein, has been demonstrated to play key roles in clearing virus-infected cells and tumour cells due to its ability of forming 'pores' on the cell membranes. Additionally, perforin is also found to be associated with human diseases such as tumours, virus infections, immune rejection and some autoimmune diseases. Until now, plenty of perforin genes have been identified in vertebrates, especially the mammals and teleost fish. Conversely, vertebrate homologue of perforin gene was not identified in the invertebrates. Although recently there have been several reviews focusing on perforin and granzymes in mammals, no one highlighted the current advances of perforin in the other vertebrates. Here, in addition to mammalian perforin, the structure, evolution, tissue distribution and function of perforin in bony fish are summarized, respectively, which will allow us to gain more insights into the perforin in lower animals and the evolution of this important pore-forming protein across vertebrates.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (β-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

Mitochondria are important not only to healthy but also dying cells. In particular, apoptotic cell death initiates when the mitochondrial outer membrane is permeabilized by Bax, a protein of the Bcl-2 family. Bax shares a structural fold with some α-helical bacterial pore-forming toxins before these proteins actively engage membranes. Despite decades of intensive research, the structures of the pores formed by these proteins are mostly unknown, mainly because the pores are assembled by different numbers of the proteins whose conformation and interaction are highly dynamic. Site-specific crosslinking of the pore-forming proteins in cellular membranes where the pores are assembled is a powerful approach to assess the biological pore structure, dynamics and function. In this chapter, we describe a cysteine-based site-specific crosslinking protocol for the Bax protein in the mitochondrial membrane. We discuss the expected results and the resulting structural-functional models for the pore-forming Bax oligomer, in comparison with other crosslinking approaches that have been used to study other mitochondrial protein complexes. At the end, we highlight the advantages of the crosslinking approaches as well as the limitations and alternative approaches.

Pore-forming proteins (PFPs) include virulence factors that are produced by many pathogenic bacteria. However, PFPs also comprise non-virulence factors, such as apoptotic Bcl2-like proteins, and also occur in non-pathogenic bacteria and indeed in all kingdoms of life. Pore-forming proteins are an ancient class of proteins, which are tremendously powerful in damaging cell membranes. In general, upon binding to lipid membranes, they convert from the soluble monomeric form into an oligomeric state, and then undergo a dramatic conformational change to form transmembrane pores. Thus, PFPs render the plasma membrane of their target cells permeable to solutes, potentially leading to cell death, or to more subtle manipulations of cellular functions. Recent cryo-EM and X-ray crystallography studies revealed high-resolution structures of several PFPs in their pre-pore and pore states, however many aspects regarding the cues that induce pore formation, the pre-pore to pore conformational transition, the mechanism of membrane permeation and associated dynamics are still less well understood, and direct visualization of the dynamics of these transitions are missing. Using high-speed atomic force microscopy (HS-AFM), the kinetics of oligomerization and the pre-pore to pore transition dynamics of various PFPs, such as Listeriolysin O (LLO), lysenin, and Perforin-2 (PFN2), could be studied. These studies revealed that LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants. In contrast, lysenin forms stable pre-pore and pore nonameric rings and HS-AFM allowed to study their diffusion on and the pH-dependent insertion into the membrane. Similarly, PFN2 underwent pre-pore to pore transition upon acidification. The openness of the HS-AFM system allowed the transition to be imaged in real time and revealed that all observed molecules transitioned into the pore state within 3s. In this chapter, we detail protocols to prepare lipids, form supported lipid bilayers, and provide guidelines for real-time, real-space HS-AFM observations of PFPs in action.

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data.

Organisms have evolved mechanisms in which cellular membranes can both be targeted and punctured thereby killing the targeted cell. One such mechanism involves the deployment of pore forming proteins (PFPs) which function by oligomerizing on cell membranes and inserting a physical pore spanning the membrane. This pore can lead to cell death by either causing osmotic flux or allowing the delivery of a secondary toxin. Pore forming proteins can be broadly classified into different families depending on the structure of the final pore; either α-PFPs using channels made from α -helices or β-PFPs using channels made from β-barrels. There are many different β-PFPs and an emerging superfamily is the aerolysin-ETX/MTX-2 superfamily. A comparison between the members of this superfamily reveals the pore forming domain is a common module yet the receptor binding region is highly variable. These structural and architectural variations lead to differences in the target recognition and determine the site of activity. Closer investigation of the topology of the family also suggests that the Toxin_10 family of PFPs could be considered as part of the aerolysin-ETX/MTX-2 superfamily. Comparatively, far less is known about how Toxin_10 proteins assemble into the final pore structure than aerolysin-ETX/MTX-2 proteins. This review aims to collate the pore forming protein members and bridge the structural similarities between the aerolysin-ETX/MTX-2 superfamily and the insecticidal Toxin_10 subfamily.

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.

Bioinformatic analyses of protein sequences play an important role in the discovery and subsequent safety assessment of insect control proteins in Genetically Modified (GM) crops. Due to the rapid adoption of high-throughput sequencing methods over the last decade, the number of protein sequences in GenBank and other public databases has increased dramatically. Many of these protein sequences are the product of whole genome sequencing efforts, coupled with automated protein sequence prediction and annotation pipelines. Published genome sequencing studies provide a rich and expanding foundation of new source organisms and proteins for insect control or other desirable traits in GM products. However, data generated by automated pipelines can also confound regulatory safety assessments that employ bioinformatics. Largely this issue does not arise due to underlying sequence, but rather its annotation or associated metadata, and the downstream integration of that data into existing repositories. Observations made during bioinformatic safety assessments are described.

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of metabolic and energetic mitochondrial dysfunction and cell stress. The most active area of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found a predominant presence of NRF-1 together with other transcription factors binding sites, involved in cell growth, proliferation, development and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as reported in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time data, it was confirmed that VDAC1 promoter activity is further stimulated when are exposed to stress. A bioinformatic survey suggested NRF-1 e HIF-1α as the most active TFBS. Their validation was obtained by mutagenesis and overexpression experiments. In conclusion, we demonstrated experimentally the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in cell stress conditions.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that βγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of βγ-CAT. The cellular action of βγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of βγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

Pore Forming Toxins (PFTs), formed mainly by virulence factors of bacteria, belongs to Pore Forming Protein (PFP) family. Secreted as soluble monomers, they bind specific targets in membranes where their oligomerization and insertion place. Lysenin, a member of the PFTs, forms and oligomer after sphingomyelin binding, the so-called prepore, which become inserted forming a pore after a conformational change triggered by a pH decrease. In crowded conditions, oligomers tends to stay in prepore form because the prepore-to-pore transition is sterically blocked. In this study, we investigate the effect of calcium ions in those crowded conditions, finding that calcium act as a trigger for lysenin insertion. We localize the residues responsible for calcium sensitivity in a small α-helix. Our results are not only one of the few complete structural descriptions of prepore-to-pore transitions but the very first that involves a calcium triggering mechanism. The presence of glutamic or aspartic acids in the insertion domains could be an indication that calcium may be a general trigger for PFTs and more generally PFP.

Pore Forming Toxins (PFTs), formed mainly by virulence factors of bacteria, belongs to Pore Forming Protein (PFP) family. Secreted as soluble monomers, they bind specific targets in membranes where their oligomerization and insertion place. Lysenin, a member of the PFTs, forms and oligomer after sphingomyelin binding, the so-called prepore, which become inserted forming a pore after a conformational change triggered by a pH decrease. In crowded conditions, oligomers tends to stay in prepore form because the prepore-to-pore transition is sterically blocked. In this study, we investigate the effect of calcium ions in those crowded conditions, finding that calcium act as a trigger for lysenin insertion. We localize the residues responsible for calcium sensitivity in a small α-helix. Our results are not only one of the few complete structural descriptions of prepore-to-pore transitions but the very first that involves a calcium triggering mechanism. The presence of glutamic or aspartic acids in the insertion domains could be an indication that calcium may be a general trigger for PFTs and more generally PFP.

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity., Physical membrane properties play a determining role in defining the sensitivity of membranes to the immune effector perforin.

Innate immunity enables host defense against pathogens and endogenous danger through inflammasomes, which are supramolecular complexes that recognize the threats and activate the immune response. Inflammasome activation often leads to pyroptosis, a highly inflammatory and lytic form of cell death, as a means of killing infected cells and releasing IL-1 family cytokines that communicate with other cells. Dysregulated inflammasome signaling results in a wide range of immune disorders including gout, sepsis, and hepatitis. Discovered as a direct killer molecule in pyroptosis, gasdermin D (GSDMD) is a pore-forming protein that represents a novel family with diverse cellular functions and pathological roles. This review summarizes current opinions in the biological mechanisms and therapeutic values of the GSDM family, particularly of GSDMD. Detailed mechanisms of auto-inhibition and pore formation by the GSDM family are presented, followed by a brief summary of the progress in the development of GSDM-targeting therapeutics.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that gd T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites.Here, we demonstrate that Plasmodium falciparum infection depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Pyroptosis, a lytic form of programmed cell death, both stimulates effective immune responses and causes tissue damage. Gasdermin (GSDM) proteins are a family of pore-forming executors of pyroptosis. While the most-studied member, GSDMD, exerts critical functions in inflammasome biology, emerging evidence demonstrates potential broad relevance for GSDM-mediated pyroptosis across diverse pathologies. In this review, we describe GSDM biology, outline conditions where inflammasomes and GSDM-mediated pyroptosis represent rational therapeutic targets, and delineate strategies to manipulate these central immunologic processes for the treatment of human disease.

Despite recent advances in signal reporter-based assays for bacteria detection and profiling, the low-cost, ultrasensitive, accurate, and fast diagnosis remains a challenge for better patient care. Herein, we present a novel bacteria identification method based on α-hemolysin-labeled sandwich assay (HLSA). A pore-forming protein, α-hemolysin, is used as an electrical signal reporter. The assay takes advantage of the specific binding of target nucleic acid with two hybridization probes: capture probe decorated magnetic microparticles and oligonucleotides detecting probe and α-hemolysin modified gold nanoparticles. α-Hemolysin was then released by competitive gold binding peptide incubation into an electric cell with a lipid bilayer between the electrodes. The nanopores formed by α-hemolysin on the lipid layer allowed target nucleic acid concentration-dependent currents for quantification. Sandwich probes against 16S rRNAs of 10 common bacteria pathogens were designed and single cell level nucleic acid concentration detection was achieved. Compared with nanopore technique-based DNA sequencing, HLSA gives a quantitive and straightforward readout that is not dependent on an ultrasensitive and expensive instrument (Axopatch 200B amplifier), thus, is faster and requires no large-scale instruments. Also, since α-hemolysin-modified nanoparticles will be washed out before the α-hemolysin releasing step without the target nucleic acid, no current will be detected, and thus, the assay is more specific. The current strategy based on the electrical signal reporter offers a new insight for pathogen and virus diagnostics.

Actinoporins are pore-forming proteins found in sea anemones. Although we now have a large collection of data on actinoporins, our knowledge is based heavily on those identified in shallow-water anemones. Because the deep sea differs considerably from shallow waters in hydrostatic pressures, temperatures, and the prey composition, the deep-sea actinoporin may have evolved in unique ways. This study, therefore, aimed to obtain new actinoporins in the deep-sea anemone Cribrinopis japonica (Actiniaria, Actiniidae). An actinoporin-like sequence was identified from the previously established C. japonica RNA-Seq database, and the complete length (663 bp) of the deep-sea actinoporin gene, Cjtox I, was obtained. In addition, a similar gene, Cjtox II (666 bp), was also identified from RNA of actinopharynx. CJTOX I and CJTOX II were similar in their primary structures, but CJTOX I lacked one residue in the middle of the protein. There was also a difference in the gene expression in live animals, where only Cjtox I was expressed in tentacles of C. japonica. In the heterologous expression where BL21 (DE3) strain was retransformed with the plasmid containing either Cjtox I or Cjtox II gene, the supernatants of both cell lysates showed hemolytic activity on the equine erythrocytes. Preincubation of the supernatants with sphingomyelin caused reduced activity, implying that the CJTOX I and II would target sphingomyelin as with other actinoporins. Because of the structures similarity to the known actinoporins and the sphingomyelin-inhibitable hemolytic activity, both CJTOX I and II were concluded to be new actinoporins, which were identified for the first time from a deep-sea anemone.

Cytotoxic lymphocytes play a key role in immune homeostasis through elimination of virally-infected and transformed target cells. They do this by employing the potent pore-forming protein, perforin, a molecule that permits cytotoxic proteases, such as granzyme B, to enter the target cell cytoplasm. The synergistic activities of perforin and granzymes bring about the destruction of target cells in a process that is now more clearly understood as a result of structural and cellular biology. These data are helping the development of new classes of immunosuppressive molecules for use in treating immune driven disease and in enhancing the success of transplant therapies. This review focuses on structural and biological aspects of perforin function.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

The malaria parasite Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2. Among them, EXP2 is thought to constitute the transmembrane pore, whereas the other components seem to play a role in unfolding the luggage proteins or providing a driving force. However, detailed functional and structural characterizations of PTEX proteins have not been performed. In this study, we expressed and characterized the membrane-associated component EXP2. Because expression of EXP2 is lethal to E. coli, EXP2 was expressed as a fusion protein with GST, and the recombinant EXP2 was obtained by protease digestion. The recombinant EXP2 formed pores in bilayer lipid membranes. The inner diameter of the pore was estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, we determined that the pore comprises approximately 10–12 EXP2 subunits. However, there is a possibility that the pore structure is different in the PTEX complex. These results provide important insights in the protein transport mechanism of PTEX, which will aid in developing new drugs targeting PTEX.

The original version of this Article contained an error in the spelling of the author Ahmed Abdelanabi, which was incorrectly given as Abdelanabi Ahmed. This has now been corrected in both the PDF and HTML versions of the Article.

Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.

Gasdermin is a recently identified family of pore-forming proteins consisting of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME), and DFNB59. Gasdermin D (GSDMD) is a downstream effector of inflammasomes, which are supramolecular complexes that activate inflammatory caspases (-1, -4, and -5 in human and -1 and -11 in mouse). GSDMD contains a functionally important N-terminal domain (GSDMD-N), a C-terminal domain, and a linker in between that is recognized and cleaved by the activated inflammatory caspases. Upon cleavage, the GSDMD-N fragments translocate on the membrane and oligomerize to form membrane-embedded pores after specifically binding to acidic lipids such as phosphatidylinositol phosphates (PIPs), phosphatidic acid (PA), phosphatidylserine (PS), and cardiolipin. The pore exhibits strong membrane-disrupting cytotoxicity in mammalian cells by disrupting the osmotic potential and also serves as a gate for extracellular release of mature IL-1β and IL-18 during pyroptosis. In this chapter, we review our current understanding of GSDM proteins in physiological and pathological cell death, with more focused discussions on its structural basis for GSDM activation and pore formation.

Glucocorticoids (GCs) play a role in stress coping by activating the glucocorticoid receptor (GR), a ligand-bound transcription factor. GCs also exert rapid effects that are nongenomic by modulating second messenger signaling, including Ca2+. However, the mechanism of action of GCs in modulating cytoplasmic free calcium level ([Ca2+]i) is unclear. We hypothesized that cortisol increases ([Ca2+]i) in zebrafish (Danio rerio) muscle, and this is independent of GR activation. Indeed, cortisol rapidly stimulated ([Ca2+]i) rise in the developing trunk muscle (DTM), and this response was not abolished in the GR knockout zebrafish. The rapid cortisol-induced ([Ca2+]i) rise was reduced with EGTA, and completely abolished by the pharmacological inhibition of the calcium release-activated calcium channel (CRACC). Also, cortisol stimulation rapidly increased the expression of Orai1, the pore forming protein subunit of CRACC, in the DTM. Altogether, rapid nongenomic action of cortisol on muscle function may involve Ca2+ signaling by CRACC gating in zebrafish.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active β-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the β-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered β-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.

More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes-ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ=4.1 nS in 1.0M KCl is moderately cation-selective (PK(+)/PCl(-)=1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r~0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300-400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids β-oxidation in intact cells but not in the corresponding lysates. The β-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the β-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of β-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal β-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis.

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a β sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the β sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.

Atomic force microscopy (AFM) is a form of contact microscopy that uses a very sharp tip to scan the surface of a sample. It provides a 3D image of the surface structure and in the force mode it can also be used to test the mechanical properties of the sample. AFM has been successfully applied to study the molecular mechanism of pore-forming proteins on model membranes. It gives information about both the structural reorganization of the membrane surface and the changes in the force required for membrane piercing upon incubation with this special type of proteins. Here we describe robust protocols to investigate the effect of pore-forming proteins in supported lipid bilayers .

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that potentiate perforin activity by applying mechanical force against the target cell. Here, we employed high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical distortion of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.One sentence summaryCytotoxic T lymphocytes use F-actin-rich protrusions at the immunological synapse to potentiate perforin-and granzyme-mediated target cell killing.

Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1β, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

For therapies targeting diseases of the gastrointestinal tract, we and others envision probiotic bacteria that synthesize and excrete biotherapeutics at disease sites. Toward this goal, we have engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide (NO), an intestinal biomarker for Crohn's disease (CD). This is accomplished by co‐expressing the pore forming protein TolAIII with the biologic, granulocyte macrophage‐colony stimulating factor (GM‐CSF). We have additionally engineered these bacteria to accumulate at sites of elevated NO by engineering their motility circuits and controlling pseudotaxis. Importantly, because we have focused on in vitro test beds, motility and biotherapeutics production are spatiotemporally characterized. Together, the targeted recognition, synthesis, and biomolecule delivery comprises a “smart” probiotics platform that may have utility in the treatment of CD. Further, this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively.