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.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.     

Granzyme B encoded by the commonly occurring human RAH allele retains pro-apoptotic activity

A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.     

Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors but also induce non-opioid excitotoxic effects. Dynorphin A can increase the intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism. In this investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A (1-13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.     

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two α/β-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating α- and β-amino acid residues, designated simply as α/β-peptide I and α/β-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of α/β-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by α/β-peptide II. The α/β-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of α/β-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. α/β-Peptide II is more effective in this regard because, unlike α/β-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although α/β-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why α/β-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of α/β-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Probing conformational changes in lipoxygenases upon membrane binding: Fine-tuning by the active site inhibitor ETYA

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Degranulation enhances presynaptic membrane packing,which protects NK cells from perforin-mediated autolysis

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.

Natural killer cells mediate largely unidirectional potent cytotoxicity against diseased cells while sparing themselves. The authors show that the NK cell membrane contains and focuses lipids of high density which shield against self-destruction, and a similar densely packed postsynaptic membrane is responsible for the perforin resistance and NK cell-mediated killing evasion of an aggressive breast cancer cell line.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state ²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using ²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic beta-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK(4) to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK(4) to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK(4) to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK(4) varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK(4) can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK(4) binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis

Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB-serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [DeltaPsim] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3-deficient cells fail to display a reduced DeltaPsim and DNA fragmentation; (b) Bax/Bak is required for optimal DeltaPsim reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced DeltaPsim and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3-deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway.     

Polyglutamine expansion in huntingtin alters its interaction with phospholipids

Huntingtin has an expanded polyglutamine tract in patients with Huntington's disease. Huntingtin localizes to intracellular and plasma membranes but the function of huntingtin at membranes is unknown. Previously we reported that exogenously expressed huntingtin bound pure phospholipids using protein-lipid overlays. Here we show that endogenous huntingtin from normal ( Hdh ^7Q/7Q) mouse brain and mutant huntingtin from Huntington's disease ( Hdh ^140Q/140Q) mouse brain bound to large unilamellar vesicles containing phosphoinositol (PI) PI 3,4-bisphosphate, PI 3,5-bisphosphate, and PI 3,4,5-triphosphate [PI(3,4,5)P3]. Huntingtin interactions with multivalent phospholipids were similar to those of dynamin. Mutant huntingtin associated more with phosphatidylethanolamine and PI(3,4,5)P3 than did wild-type huntingtin, and associated with other phospholipids not recognized by wild-type huntingtin. Wild-type and mutant huntingtin also bound to large unilamellar vesicles containing cardiolipin, a phospholipid specific to mitochondrial membranes. Maximal huntingtin-phospholipid association required inclusion of huntingtin amino acids 171–287. Endogenous huntingtin recruited to the plasma membrane in cells that incorporated exogenous PI 3,4-bisphosphate and PI(3,4,5)P3 or were stimulated by platelet-derived growth factor or insulin growth factor 1, which both activate PI 3-kinase. These data suggest that huntingtin interacts with membranes through specific phospholipid associations and that mutant huntingtin may disrupt membrane trafficking and signaling at membranes.     

Behavior of giant vesicles with anchored DNA molecules

We study changes in curvature and elastic properties of lipid membranes induced by anchoring of long hydrophilic polymers at low polymer surface concentrations (corresponding to the mushroom regime). The effect of anchored polymers on the membrane spontaneous curvature is characterized by monitoring the changes in the fluctuation spectra and the morphology of giant unilamellar vesicles. The polymers used in our study are fluorescently labeled and biotinylated lambda-phage DNA molecules which bind to biotinylated giant unilamellar vesicles via a biotin-avidin-biotin linkage. By varying the amount of biotinylated lipid in the membrane, we control the surface concentration of anchors. At low anchor concentrations, the spontaneous curvature of the membrane increases linearly with the DNA concentration. The linear increase is consistent with theoretical predictions for polymer surface concentrations in the mushroom regime. At higher anchor concentrations, which should still belong to the mushroom regime, the vesicles undergo budding transitions. In this latter regime, the bud size is used to estimate the polymer-induced membrane curvature.     

Affinity driven molecular transfer from erythrocyte membrane to target cells

A wide variety of antimicrobial peptides are known to bind to - and disrupt microbial plasma membranes. Recently, derivatives of the antimicrobial peptide dermaseptin S4 were shown to selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocyte parasite. In this study, we present evidence in support of the ability of a membrane-bound peptide, the dermaseptin S4 derivative K(4)-S4(1-13)a, to transfer from red blood cells (RBCs) to another distant membrane. Binding of K(4)-S4(1-13)a to the plasma membrane of RBCs was assessed in vitro and in vivo, and found to be rapid, spontaneous and receptor independent, as was the transfer of the RBC-bound peptide to the plasma membrane of microorganisms. The present study further provides a basis for the possible use of RBCs as a transport vehicle to deliver drugs to distant targets. This drug delivery system involves the transient "loading" of RBCs with a lipophilic "hook" peptide. Such a peptide has enough affinity for the RBC's plasma membrane to bind to the membrane, but given the opportunity, the peptide will exit its position and transfer to another (target) cell for which it has a greater affinity. The efficacy of such an affinity driven transfer system was demonstrated experimentally by the transfer of K(4)-S4(1-13)a from pre-loaded RBCs to bacteria, yeast and protozoan target cells.     

Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins

Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, ³¹P-NMR, electron paramagnetic resonance (EPR), proton NMR (¹H-NMR), deuterium NMR (²H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.     

Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin

Human granzyme B (GrB) released from cytotoxic lymphocytes plays a key role in the induction of target cell apoptosis when internalized in the presence of perforin. Here we demonstrate that GrB also possesses a potent extracellular matrix remodeling activity. Both native and recombinant GrB caused detachment of immortalized and transformed cell lines, primary endothelial cells, and chondrocytes. Cell detachment by GrB induced endothelial cell death (anoikis). GrB also inhibited tumor cell spreading, migration, and invasion in vitro. Investigation into the underlying mechanism revealed that GrB efficiently cleaves three proteins involved in extracellular matrix structure and function: vitronectin, fibronectin, and laminin. In vitronectin, GrB cleaves after an Arg-Lys-Asp (RGD) motif, which is part of the integrin-binding site found in matrix proteins. We propose that targeting of the integrin-extracellular matrix interface by GrB may allow perforin-independent killing of target cells via anoikis, restrict motility of tumor cells, facilitate lymphocyte migration, or directly reduce virus infectivity. It may also contribute to tissue destruction in diseases in which extracellular GrB is evident, such as rheumatoid arthritis and atherosclerosis.     

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 °C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 °C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 °C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.