Intercellular communication via the endo-lysosomal system: Translocation of granzymes through membrane barriers

Cytotoxic lymphocytes (CLs) are responsible for the clearance of virally infected or neoplastic cells. CLs possess specialised lysosome-related organelles called granules which contain the granzyme family of serine proteases and perforin. Granzymes may induce apoptosis in the target cell when delivered by the pore forming protein, perforin. Here we follow the perforin-granzyme pathway from synthesis and storage in the granule, to exocytosis and finally delivery into the target cell. This review focuses on the controversial subject of perforin-mediated translocation of granzymes into the target cell cytoplasm. It remains unclear whether this occurs at the cell surface with granzymes moving through a perforin pore in the plasma membrane, or if it involves internalisation of perforin and granzymes and subsequent release from an endocytic compartment. The latter mechanism would represent an example of cross talk between the endo-lysosomal pathways of individual cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Capturing pore-forming intermediates of MACPF and binary toxin assemblies by cryoEM

Deployed by both pathogenic bacteria and host immune systems, pore-forming proteins rupture target membranes and can serve as conduits for effector proteins. Understanding how these proteins work relies on capturing assembly intermediates. Advances in cryoEM allowing in silico purification of heterogeneous assemblies has led to new insights into two main classes of pore-forming proteins: membrane attack complex perforin (MACPF) proteins and binary toxins. The structure of an immune activation complex, sMAC, shows how pores form by sequential templating and insertion of β-hairpins. CryoEM structures of bacterial binary toxins present a series of transitions along the pore formation pathway and reveal a general mechanism of effector protein translocation. Future developments in time-resolved cryoEM could capture and place short-lived states along the trajectory of pore-formation.     

Involvement of granule proteins in T-cell-mediated cytolysis

Cytolytic T lymphocytes (CTL) and large granular lymphocytes contain dense cytoplasmic granules which, when isolated, are lytic for a variety of target cells. Granule proteins are released from the effector cell upon target cell interaction, further suggesting that they play a role in the cytolytic mechanism. Major proteins in CTL granules are a family of serine esterases (granzymes) and a pore-forming protein called perforin (cytolysin). Despite structural similarities between functionally conserved regions of perforin and the ninth component of complement (C9), these two lytic molecules are clearly distinct in their mode of target cell recognition. Perforin, unlike C9, is not dependent on a protein receptor molecule but binds to the target cell membrane via phosphorylcholine in a Ca2(+)-dependent manner. Here, we discuss the stimulus-secretion model for T-cell-mediated cytotoxicity with respect to our current understanding of perforin and the granzyme proteases.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.     

Granzyme M: behind enemy lines

The granule-exocytosis pathway is the major mechanism via which cytotoxic lymphocytes eliminate virus-infected and tumor cells. In this pathway, cytotoxic lymphocytes release granules containing the pore-forming protein perforin and a family of serine proteases known as granzymes into the immunological synapse. Pore-formation by perforin facilitates entry of granzymes into the target cell, where they can activate various (death) pathways. Humans express five different granzymes, of which granzymes A and B have been most extensively characterized. However, much less is known about granzyme M (GrM). Recently, structural analysis and advanced proteomics approaches have determined the primary and extended specificity of GrM. GrM functions have expanded over the past few years: not only can GrM efficiently induce cell death in tumor cells, it can also inhibit cytomegalovirus replication in a noncytotoxic manner. Finally, a role for GrM in lipopolysaccharide-induced inflammatory responses has been proposed. In this review, we recapitulate the current status of GrM expression, substrate specificity, functions, and inhibitors.     

Perforin rapidly induces plasma membrane phospholipid flip-flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.     

Disparate proteins use similar architectures to damage membranes

Membrane disruption can efficiently alter cellular function; indeed, pore-forming toxins (PFTs) are well known as important bacterial virulence factors. However, recent data have revealed that structures similar to those found in PFTs are found in membrane active proteins across disparate phyla. Many similarities can be identified only at the 3D-structural level. Of note, domains found in membrane-attack complex proteins of complement and perforin (MACPF) resemble cholesterol-dependent cytolysins from Gram-positive bacteria, and the Bcl family of apoptosis regulators share similar architectures with Escherichia coli pore-forming colicins. These and other correlations provide considerable help in understanding the structural requirements for membrane binding and pore formation.     

Design Parameters for Granzyme-Mediated Cytotoxic Lymphocyte Target-Cell Killing and Specificity

Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.     

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding

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.     

Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Perforin-mediated target-cell death and immune homeostasis

The granule exocytosis pathway of cytotoxic lymphocytes is crucial for immune surveillance and homeostasis. The trafficking of granule components, including the membrane-disruptive protein perforin, to the immunological synapse leads to the delivery of granule proteases (granzymes) into the target cell and its destruction through apoptosis. Several independent molecular abnormalities associated with defects of either granule trafficking or perforin function can cause cytotoxic lymphocyte dysfunction. In humans, inherited perforin mutations result in severe immune dysregulation that manifests as familial haemophagocytic lymphohistiocytosis. This Review describes recent progress in defining the structure, function, biochemistry and cell biology of perforin.     

All human granzymes target hnRNP K that is essential for tumor cell viability

Granule exocytosis by cytotoxic lymphocytes is the key mechanism to eliminate virus-infected cells and tumor cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes. All human granzymes display distinct substrate specificities and induce cell death by cleaving critical intracellular death substrates. In the present study, we show that all human granzymes directly cleaved the DNA/RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K), designating hnRNP K as the first known pan-granzyme substrate. Cleavage of hnRNP K was more efficient in the presence of RNA and occurred in two apparent proteolysis-sensitive amino acid regions, thereby dissecting the functional DNA/RNA-binding hnRNP K domains. HnRNP K was cleaved under physiological conditions when purified granzymes were delivered into living tumor cells and during lymphokine-activated killer cell-mediated attack. HnRNP K is essential for tumor cell viability, since knockdown of hnRNP K resulted in spontaneous tumor cell apoptosis with caspase activation and reactive oxygen species production. This apoptosis was more pronounced at low tumor cell density where hnRNP K knockdown also triggered a caspase-independent apoptotic pathway. This suggests that hnRNP K promotes tumor cell survival in the absence of cell-cell contact. Silencing of hnRNP K protein expression rendered tumor cells more susceptible to cellular cytotoxicity. We conclude that hnRNP K is indispensable for tumor cell viability and our data suggest that targeting of hnRNP K by granzymes contributes to or reinforces the cell death mechanisms by which cytotoxic lymphocytes eliminate tumor cells.     

Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.     

Permeability characteristics of cell-membrane pores induced by ostreolysin A/pleurotolysin B,binary pore-forming proteins from the oyster mushroom

Proteins from the oyster mushroom, 15 kDa ostreolysin A (OlyA), and 59 kDa pleurotolysin B (PlyB) with a membrane attack complex/perforin (MACPF) domain, damage cell membranes as a binary cytolytic pore-forming complex. Measurements of single-channel conductance and transmembrane macroscopic current reveal that OlyA/PlyB form non-selective ion-conducting pores with broad, skewed conductance distributions in N18 neuroblastoma and CHO-K1 cell membranes. Polyethylene-glycol 8000 (hydrodynamic radius of 3.78 nm) provides almost complete osmotic protection against haemolysis, which strongly suggests a colloid-osmotic type of erythrocyte lysis. Our data indicate that OlyA/PlyB form transmembrane pores of varied sizes, as other pore-forming proteins with a MACPF domain.     

Functional Dissection of Toxoplasma gondii Perforin-like Protein 1 Reveals a Dual Domain Mode of Membrane Binding for Cytolysis and Parasite Egress

The recently discovered role of a perforin-like protein (PLP1) for rapid host cell egress by the protozoan parasite Toxoplasma gondii expanded the functional diversity of pore-forming proteins. Whereas PLP1 was found to be necessary for rapid egress and pathogenesis, the sufficiency for and mechanism of membrane attack were yet unknown. Here we further dissected the PLP1 knock-out phenotype, the mechanism of PLP1 pore formation, and the role of each domain by genetic complementation. We found that PLP1 is sufficient for membrane disruption and has a conserved mechanism of pore formation through target membrane binding and oligomerization to form large, multimeric membrane-embedded complexes. The highly conserved, central MACPF domain and the β-sheet-rich C-terminal domain were required for activity. Loss of the unique N-terminal extension reduced lytic activity and led to a delay in rapid egress, but did not significantly decrease virulence, suggesting that small amounts of lytic activity are sufficient for pathogenesis. We found that both N- and C-terminal domains have membrane binding activity, with the C-terminal domain being critical for function. This dual mode of membrane association may promote PLP1 activity and parasite egress in the diverse cell types in which this parasite replicates.     

EGFR-Targeted Granzyme B Expressed in NK Cells Enhances Natural Cytotoxicity and Mediates Specific Killing of Tumor Cells

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells'' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.     

Granzymes drive a rapid listeriolysin O-induced T cell apoptosis

The Listeria monocytogenes protein listeriolysin O (LLO) is a pore-forming protein essential for virulence. Although the major role for LLO is to allow L. monocytogenes entry into the cytosol, it also induces apoptosis of activated lymphocytes, an obligatory cellular response that modulates the infection. Induction of apoptosis by LLO proceeds through a fast, caspase-dependent pathway and a slow, caspase-independent pathway. Polyclonal T cell lines were generated from either normal mice or mice deficient in granzyme and perforin proteins, and then treated with apoptogenic doses of LLO. In this study we show that apoptosis of lymphocytes induced by LLO was characterized by activation of caspases as quickly as 30 min that was dependent on the expression of granzymes. In the absence of granzymes, all parameters of apoptosis such as caspase activation, phosphatidylserine exposure, mitochondrial depolarization, and DNA fragmentation were dramatically reduced in magnitude. Removal of perforin inhibited the apoptotic effect of LLO on cells by approximately 50%. Neutralization of intracellular acidification using chloroquine inhibited the rapid apoptotic death. In agreement with these findings granzyme-deficient mice harbored lower bacterial titers and decrease splenic pathology compared with normal mice following L. monocytogenes infection. Thus, LLO exploits apoptotic enzymes of the adaptive immune response to eliminate immune cells and increase its virulence.     

Cytotoxic T lymphocyte-induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis

Cytotoxic T lymphocyte (CTL)-induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB(-/-) CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB(-/-) CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB(-/-) mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that assemble on bacterial membranes to form a porelike structure referred to as the “membrane attack complex” (MAC). C8 contains three genetically distinct subunits (C8α, C8β, C8γ) arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. C6, C7 C8α, C8β, and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8γ subunit is unrelated and belongs to the lipocalin family of proteins that display a β-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8α MACPF domain were recently reported and both display a fold similar to those of the bacterial pore-forming cholesterol-dependent cytolysins (CDCs). In the present study, we determined the crystal structure of the human C8α MACPF domain disulfide-linked to C8γ (αMACPF-γ) at 2.15 Å resolution. The αMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8γ. One is in a previously characterized 19-residue insertion (indel) in C8α and fills the entrance to the putative C8γ ligand-binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8γ β-barrel. The latter interaction induces conformational changes in αMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X₆-G-G in αMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.