Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition

Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.     

Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins

Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover.     

Recognition of cisplatin adducts by cellular proteins

Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival.     

Recognition of damaged regions in DNA by oligopeptides and proteins

The binding of various damaged DNAs to the single-strand binding protein coded for by gene 32 from bacteriophage T4, on the one hand, and of oligopeptides containing tryptophan and lysine residues, on the other hand, is described. These molecules exhibit a higher affinity for modified DNA than for native DNA in so far as modification results in a local destabilization of the double-stranded structure of the nucleic acid. Stacking interactions between aromatic amino acids and nucleic acid bases appear to play a crucial role in the recognition of destabilized regions induced by chemical agents (carcinogens and antitumor drugs). These interactions confer to the peptide lysyl-tryptophyl-lysine an endonucleolytic activity specific for apurinic sites. From results obtained with such oligopeptides a model for the active sites of Ap-endonucleases is proposed which could account for the strategy used by the denV endonuclease from phage T4 during the first step of excision repair of pyrimidine dimers in DNA. The effect of the overall conformation of modified DNA on repair efficiency is discussed.     

On the mechanism of sphingomyelin interaction with solubilized membrane proteins

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. FC epsilon-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two- to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in FC epsilon-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphingomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., FC epsilon R.     

Recognition of influenza virus proteins by cytotoxic T lymphocytes

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.     

Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes.

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.     

Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm⁺), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg²⁺, Mn²⁺ and Co²⁺ and the cleavage activity was suppressed by Zn²⁺. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.     

Oligomerization and pore formation of a sphingomyelin-specific toxin,lysenin

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of approximately 3 nm. Antibody scanning analysis suggested that the C-terminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.     

Bi-stability, hysteresis, and memory of voltage-gated lysenin channels

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages. We report on the consequences of slow voltage-induced gating of lysenin channels inserted into a planar Bilayer Lipid Membrane (BLM), and demonstrate that these pore-forming proteins constitute memory elements that manifest gating bi-stability in response to variable external voltages. The hysteresis in macroscopic currents dynamically changes when the time scale of the voltage variation is smaller or comparable to the characteristic conformational equilibration time, and unexpectedly persists for extremely slow-changing external voltage stimuli. The assay performed on a single lysenin channel reveals that hysteresis is a fundamental feature of the individual channel unit and an intrinsic component of the gating mechanism. The investigation conducted at different temperatures reveals a thermally stable reopening process, suggesting that major changes in the energy landscape and kinetics diagram accompany the conformational transitions of the channels. Our work offers new insights on the dynamics of pore-forming proteins and provides an understanding of how channel proteins may form an immediate record of the molecular history which then determines their future response to various stimuli. Such new functionalities may uncover a link between molecular events and macroscopic processing and transmission of information in cells, and may lead to applications such as high density biologically-compatible memories and learning networks.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus

The hydrolysis of d-erythro beef brain sphingomyelin and $\text{d,l}\text{-erythro}\text{-N-}\text{palmitoylsphingomyelin}$ dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30°C for beef brain sphingomyelin and 41°C for N-palmitoylsphingosinephosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus.

The hydrolysis of D-erythro beef brain sphingomyelin and D,L-erythro-N-palmitoylsphingomyelin dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30 degrees C for beef brain sphingomyelin and 41 degrees C for N-palmitoylsphingosine-phosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase-transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

Recognition of super-secondary structure in proteins

A procedure to recognize super-secondary structure in protein sequences is described. An idealized template, derived from known super-secondary structures, is used to locate probable sites by matching with secondary structure probability profiles. We applied the method to the identification of βαβ units in β/α type proteins with 75% accuracy. The location of super-secondary structure was then used to refine the original (Garnier et al., 1978) secondary structure prediction resulting in an 8.8% improvement, which correctly assigned 83% of secondary structure elements in 14 proteins. Slight modifications to the Garnier et al. method arc suggested, producing a more accurate identification of protein class and a better prediction for β/α. type proteins. A method for the incorporation of hydrophobic information into the prediction is also described.     

Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin

Amyloid beta peptide (Abeta) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Abeta and of the amyloid precursor protein (APP) is unknown. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Abeta42 directly activates neutral SMase and downregulates SM levels, whereas Abeta40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered Abeta40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).     

Interbilayer interactions between sphingomyelin and sphingomyelin/cholesterol bilayers.

Pressure versus fluid spacing relations have been obtained for sphingomyelin bilayers in the gel phase and equimolar sphingomyelin/cholesterol in the liquid-crystalline phase by the use of X-ray diffraction analysis of osmotically stressed aqueous dispersions and oriented multilayers. For interbilayer separations in the range of 5-20 A, the repulsive hydration pressure decays exponentially with increasing fluid spacing. The decay length (lambda) of this repulsive pressure is about 2 A for both bovine brain and N-tetracosanoylsphingomyelin, similar to that previously found for phosphatidylcholine bilayers. However, both the magnitude of the hydration pressure and the magnitude of the dipole potential (V) measured for monolayers in equilibrium with liposomes are considerably smaller for sphingomyelin than for either gel or liquid-crystalline phosphatidylcholine bilayers. Addition of equimolar cholesterol increases both the magnitude of the hydration pressure and the dipole potential. These data suggest that the magnitude of the hydration pressure depends on the electric field at the interface as given by (V/lambda)2. For sphingomyelin bilayers, there is a sharp upward break in the pressure-fluid spacing relation at an interbilayer spacing of about 5 A, indicating the onset of steric hindrance between the head groups of apposing bilayers.     

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels

Background

The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate.

Results

The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO₂ NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels.

Conclusion

We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.