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The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains unclear. Here, we demonstrate that survival motor neuron (SMN) protein—the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)—binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5′ untranslated region (UTR) and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins that are involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs encoding proteins that are relevant to SMA pathogenesis.

In this issue of Cell Reports Methods, Sadi et al. present a nuclear magnetic resonance approach for quantitative assessment of protein interactions with lipid membranes. It is sensitive, applicable to diverse membrane systems, covers a broad range of KDs, and does not require large amounts of material.

Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity (KDapp. 0.6 μM) compared to the parent peptide. When coupled to paramagnetic beads or a chromatographic matrix, the optimized ligand was shown to selectively enrich antibodies from complex protein mixtures.

The fungal genus Aspergillusis of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. We have generated genome sequences for ten novel, highly diverse Aspergillusspecies and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina.Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-15N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.

Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.

Equinatoxin II is a pore-forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal α-helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however, the role of this specificity is unknown. On the other hand, sphingomyelin is considered an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here, we used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid-ordered phase over the liquid-disordered one and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhances membrane binding of the toxin but is not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid-ordered and -disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen–Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to γ and ε isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4mol %) and the individual bilayer lipids was studied by 31P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20–40nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state 31P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The 2H and 31P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membrane-embedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp45, EqtII Trp116/117 and EqtII Trp149, were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp116/117 resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp45 and EqtII Trp149, was altered to a certain extent. EqtII Trp149 was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with 2H2O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp45 and EqtII Trp149 were more exposed to water, and also remained as such in the membrane-bound form.

Equinatoxin II (EqtII) is a cysteinless pore-forming protein from sea anemoneActinia equina.Three cysteine mutants were produced in anE. coliexpression system in order to study the topology of lysine 77, arginine 126, and alanine 179. Accessibility of an introduced thiol group in the water soluble mutants was studied by using the thiol specific reagent fluorescein maleimide. In aqueous solution all three mutants were readily modified with the probe, indicating their accessibility to the solvent. Mutants were also biotinylated with biotin maleimide, enabling coupling with avidin–fluorescein isothiocyanate (avidin-FITC). After binding and insertion of biotinylated toxins into liposomes, avidin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intra- or extravesicularly located thiols. All the mutated residues are found to be located on the outside of the lipid vesicles. The results proved the biotin-avidin system as suitable for topological studies of proteins creating pores in membranes.

Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemoneActinia equina.Its mode of action is still poorly understood. In order to initiate further structure–function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure. The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII. The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed inEscherichia coli.Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system. Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.

Crystal structures of the eukaryotic pore-forming toxin sticholysin II, its complex with phosphocholine, and electron microscopy of 2D crystals on lipid monolayer give insights into the sequential steps of the actinoporin membrane penetration.