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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., (Copyright © 2020 Bayly-Jones, Pang, Spicer, Whisstock and Dunstone.)

The pore forming, Ca 2+ -dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca 2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca 2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin's cytotoxic function. These results revealed an unexpected role for the Ca 2+ -dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.

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., (Copyright © 2017. Published by Elsevier Ltd.)

Perforin is a highly cytotoxic pore-forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca 2+ -rich endoplasmic reticulum, remains unknown. Here, we show that N-linked glycosylation of the perforin C-terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C-terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C-terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post-translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule., (© 2017 The Authors.)

Pore Forming Toxins (PFTs) represent a key mechanism for permitting the passage of proteins and small molecules across the lipid membrane. These proteins are typically produced as soluble monomers that self-assemble into ring-like oligomeric structures on the membrane surface. Following such assembly PFTs undergo a remarkable conformational change to insert into the lipid membrane. While many different protein families have independently evolved such ability, members of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) superfamily form distinctive giant β-barrel pores comprised of up to 50 monomers and up to 300Å in diameter. In this review we focus on recent advances in understanding the structure of these giant MACPF/CDC pores as well as the underlying molecular mechanisms leading to their formation. Commonalities and evolved variations of the pore forming mechanism across the superfamily are discussed. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca(2+) and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca(2+) ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca(2+) binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin–granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology.

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here we characterize the origins and evolution of perforin., Results: We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1 gene, and that it shares a common ancestor with the functionally related complement proteins., Conclusions: The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism.

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The secretory granule-mediated cell death pathway is the key mechanism for elimination of virus-infected and transformed target cells by cytotoxic lymphocytes. The formation of the immunological synapse between an effector and a target cell leads to exocytic trafficking of the secretory granules and the release of their contents, which include pro-apoptotic serine proteases, granzymes, and pore-forming perforin into the synapse. There, perforin polymerizes and forms a transmembrane pore that allows the delivery of granzymes into the cytosol, where they initiate various apoptotic death pathways. Unlike relatively redundant individual granzymes, functional perforin is absolutely essential for cytotoxic lymphocyte function and immune regulation in the host. Nevertheless, perforin is still the least studied and understood cytotoxic molecule in the immune system. In this review, we discuss the current state of affairs in the perforin field: the protein's structure and function as well as its role in immune-mediated diseases.

The pore-forming protein perforin is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, we examined perforin function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their perforin (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast, truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perforin-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perforin function is a predictor of FHL severity.

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.

Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane beta-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development.

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen. [ABSTRACT FROM AUTHOR]

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca2+ -dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca2+ binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforinlike) domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation. [ABSTRACT FROM AUTHOR]

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands. [Display omitted] • A fragment screen was used to identify binders of the cytolytic protein perforin. • Structure-guided design enabled optimisation to yield ligands with nanomolar potency in cells. • Target engagement of ligands with perforin was demonstrated using 19F NMR and SPR. • These ligands have superior physicochemical properties compared to previous inhibitors. • Further cellular studies will be undertaken to define the mechanism of action of these novel ligands. [ABSTRACT FROM AUTHOR]