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Transcriptomic profiling of venom producing tissues from different animals is an effective approach for discovering new toxins useful in biotechnological and pharmaceutical applications, as well in evolutionary comparative studies of venomous animals. Stichodactyla helianthus is a Caribbean sea anemone which produces actinoporins as part of its toxic venom. This family of pore forming toxins is multigenic and at least two different isoforms, encoded by separate genes, are produced by S. helianthus . These isoforms, sticholysins I and II, share 93% amino acid identity but differ in their pore forming activity and act synergistically. This observation suggests that other actinoporin isoforms, if present in the venomous mixture, could offer an advantageous strategy to modulate whole venom activity. Using high-throughput sequencing we generated a de novo transcriptome of S. helianthus and determined the relative expression of assembled transcripts using RNA-Seq to better characterize components of this species' venom, focusing on actinoporin diversity. Applying this approach, we have discovered at least one new actinoporin variant from S. helianthus in addition to several other putative venom components. [ABSTRACT FROM AUTHOR]

Abstract: Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH2-terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid–protein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH2-terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these five basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein–vesicle and protein–protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers. [Copyright &y& Elsevier]

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP ( Anti fungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases. [ABSTRACT FROM AUTHOR]

Botrytis blight (gray mold), caused by Botrytis cinema, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic lowmolecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinema isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinema. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases. [ABSTRACT FROM AUTHOR]

The mechanics of the extracellular matrix (ECM) determine cell activity and fate through mechanoresponsive proteins including Yes-associated protein 1 (YAP). Rigidity and viscous relaxation have emerged as the main mechanical properties of the ECM steering cell behavior. However, how cells integrate coexisting ECM rigidity and viscosity cues remains poorly understood, particularly in the high-stiffness regime. Here, we have exploited engineered stiff viscoelastic protein hydrogels to show that, contrary to current models of cell-ECM interaction, substrate viscous energy dissipation attenuates mechanosensing even when cells are exposed to higher effective rigidity. This unexpected behavior is however readily captured by a pull-and-hold model of molecular clutch-based cell mechanosensing, which also recapitulates opposite cellular response at low rigidities. Consistent with predictions of the pull-and-hold model, we find that myosin inhibition can boost mechanosensing on cells cultured on dissipative matrices. Together, our work provides general mechanistic understanding on how cells respond to the viscoelastic properties of the ECM. [ABSTRACT FROM AUTHOR]

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane. [ABSTRACT FROM AUTHOR]

Toxins have been thoroughly studied for their use as therapeutic agents in search of an improvement in toxic efficiency together with a minimization of their undesired side effects. Different studies have shown how toxins can follow different intracellular pathways which are connected with their cytotoxic action inside the cells. The work herein presented describes the different pathways followed by the ribotoxin α -sarcin and the fungal RNase T1, as toxic domains of immunoconjugates with identical binding domain, the single chain variable fragment of a monoclonal antibody raised against the glycoprotein A33. According to the results obtained both immunoconjugates enter the cells via early endosomes and, while α -sarcin can translocate directly into the cytosol to exert its deathly action, RNase T1 follows a pathway that involves lysosomes and the Golgi apparatus. These facts contribute to explaining the different cytotoxicity observed against their targeted cells, and reveal how the innate properties of the toxic domain, apart from its catalytic features, can be a key factor to be considered for immunotoxin optimization. [ABSTRACT FROM AUTHOR]

Spanish or Spanish-speaking scientists represent a remarkably populated group within the scientific community studying pore-forming proteins. Some of these scientists, ourselves included, focus on the study of actinoporins, a fascinating group of metamorphic pore-forming proteins produced within the venom of several sea anemones. These toxic proteins can spontaneously transit from a water-soluble fold to an integral membrane ensemble because they specifically recognize sphingomyelin in the membrane. Once they bind to the bilayer, they subsequently oligomerize into a pore that triggers cell-death by osmotic shock. In addition to sphingomyelin, some actinoporins are especially sensible to some other membrane components such as cholesterol. Our group from Universidad Complutense of Madrid has focused greatly on the role played by sterols in this water–membrane transition, a question which still remains only partially solved and constitutes the main core of the article below. [ABSTRACT FROM AUTHOR]

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed. [ABSTRACT FROM AUTHOR]

Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal β-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane. [Display omitted] • Fungal ribotoxin α-sarcin has been shown to aggregate liposomes of complex composition. • This ability of α-sarcin is influenced by the lipid headgroups and the degree of saturation of the acyl chains. • α-sarcin is particularly sensitive to the negative charge density on the membrane surface, also affected by cholesterol. • α-sarcin requires shorter acyl chains to be able to induce lipid mixing between the aggregated liposomes. [ABSTRACT FROM AUTHOR]

Filamentous fungi are an invaluable source for biocontrol strategies and for production and development of different antifungal polypeptides. Within this context, cysteine-rich antifungal AFP-like peptides stand out among many different antimicrobial compounds given their production easiness, stability, versatility, and efficacy. AFP from Aspergillus giganteus represents the hallmark of this still increasing family of antifungal polypeptides. Close in silico analyses of the Fusarium graminearum genome revealed the presence of an AFP-like peptide, here designated as FgAFP. This new peptide was cloned, produced in the yeast Pichia pastoris , and characterized. The results obtained showed its strong and specific antifungal activity against several well-recognized maize pathogens, but inefficacy against F. oxysporum , which has not been described as a natural biological competitor of other fungal pathogens assayed. All results together suggest that this small peptide is an important factor for the fungal interplays involved in maize infection and reveals unforeseen potential biotechnological applications for FgAFP in maize production and storage. [ABSTRACT FROM AUTHOR]

Sticholysins I and II (StnI and StnII), α-pore forming toxins from the sea anemone Stichodactyla helianthus, are water-soluble toxic proteins which upon interaction with lipid membranes of specific composition bind to the bilayer, extend and insert their N-terminal α-helix, and become oligomeric integral membrane structures. The result is a pore that leads to cell death by osmotic shock. StnI and StnII show 93% of sequence identity, but also different membrane pore-forming activities. The hydrophobicity profile along the first 18 residues revealed differences which were canceled by substituting StnI amino acids 2 and 9. Accordingly, the StnID9A mutant, and the corresponding StnIE2AD9A variant, showed enhanced hemolytic activity. They also revealed a key role for an exposed salt bridge between Asp9 and Lys68. This interaction is not possible in StnII but appears conserved in the other two well-characterized actinoporins, equinatoxin II and fragaceatoxin C. The StnII mutant A8D showed that this single replacement was enough to transform StnII into a version with impaired pore-forming activity. Overall, the results show the key importance of this salt bridge linking the N-terminal stretch to the β-sandwich core. A conclusion of general application for the understanding of salt bridges role in protein design, folding and stability. [ABSTRACT FROM AUTHOR]

In this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using dimonounsaturated phosphatidylcholine (PC) analogues (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUVs) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn-LUV interaction, we observed that the bilayer affinity constant (Ka) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins' N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC. [ABSTRACT FROM AUTHOR]

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae , respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins. [ABSTRACT FROM AUTHOR]

During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central β-turn resembling that of its yeast counterpart. Disruption of the-fold by D-alanine substitution of the conserved central Gly6-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central β-turn but instead required two conserved Trp¹-Cys² residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division. [ABSTRACT FROM AUTHOR]

Actinoporins are pore-forming toxins produced by sea anemones. They exert their activity by binding to the membranes of target cells. There, they oligomerize, forming cation-selective pores, and inducing cell death by osmotic shock. In the early days of the field, it was shown that accessible sphingomyelin (SM) in the bilayer is required for the activity of actinoporins. While these toxins can also act on membranes composed solely of phosphatidylcholine (PC) with a high amount of cholesterol (Chol), consensus is that SM acts as a lipid receptor for actinoporins. It has been shown that the 2NH and 3OH moieties of SM are essential for actinoporin recognition. Hence, we wondered if ceramide-phosphoethanolamine (CPE) could also be recognized. Like SM, CPE has the 2NH and 3OH groups, and a positively charged headgroup. While actinoporins have been observed to affect membranes containing CPE, Chol was always also present, with the recognition of CPE remaining unclear. To test this possibility, we used sticholysins, produced by the Caribbean Sea anemone Stichodactyla helianthus. Our results show that sticholysins can induce calcein release on vesicles composed only of PC and CPE, in absence of Chol, in a way that is comparable to that induced on PC:SM membranes. [ABSTRACT FROM AUTHOR]

And by UCM-Banco Santander Grants PR75/18-21561, PR87/19-22556, and PR108/20-26896 (to A.M.-d.-P.)." now reads: "This research was supported by the Juselius Foundation (J.P.S. and J.P.-O.) And by UCM-Banco Santander Grants PR75/18-21561, PR87/19-22556, PR108/20-26896 and a REACT-EU grant from the Comunidad de Madrid to the ANTICIPA prorogued project of Complutense University of Madrid (to A.M.-d.-P.).". [Extracted from the article]

Fungi establish a complex network of biological interactions with other organisms in nature. Inmany cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study howribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies. [ABSTRACT FROM AUTHOR]

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against ribosomes and suggested as potential insecticides. Here we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are of interest. [ABSTRACT FROM AUTHOR]

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are the pore-forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families that give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here, using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other's activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores because (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help us understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity. [ABSTRACT FROM AUTHOR]

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans Δ 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs. [ABSTRACT FROM AUTHOR]

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger, with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties. Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently, four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity, being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells. [ABSTRACT FROM AUTHOR]

Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3ΔW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking. This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate ( IMTXA33HtA3ΔW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickel-nitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3ΔW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients. [ABSTRACT FROM AUTHOR]

The pore forming capacity of Sticholysin II (StnII; isolated from Stichodactyla helianthus ) in bilayer membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoylsphingomyelin (PSM) and either cholesterol or palmitoyl ceramide (PCer) has been examined. The aim of the study was to elucidate how the presence of differently ordered PSM domains affected StnII oligomerization and pore formation. Cholesterol is known to enhance pore formation by StnII, and our results confirmed this and provide kinetic information for the process. The effect of cholesterol on bilayer permeabilization kinetics was concentration-dependent. In the concentration regime used (2.5–10 nmol cholesterol in POPC:PSM 80:20 by nmol), cholesterol also increased the acyl chain order in the fluid PSM domain and thus decreased bilayer fluidity, suggesting that fluidity per se was not responsible for cholesterol's effect. Addition of PCer (2.5–10 nmol) to the POPC:PSM (80:20 by nmol) bilayers attenuated StnII-induced pore formation, again in a concentration-dependent fashion. This addition also led to the formation of a PCer-rich gel phase. Addition of cholesterol to PCer-containing membranes could partially reduce the inhibitory effect of PCer on StnII pore formation. We conclude that the physical state of PSM (as influenced by either cholesterol or PCer) affected StnII binding and pore formation under the conditions examined. [ABSTRACT FROM AUTHOR]

Ribotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with α-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of α-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells. [ABSTRACT FROM AUTHOR]

Sea anemone actinoporins constitute an optimum model to investigate mechanisms of membrane pore formation. All actinoporins of known structure show a general fold of a β-sandwich motif flanked by two α-helices. The crucial structure for pore formation seems to be the helix located at the N-terminal end. The role of several other protein regions in membrane attachment is also well established. However, not much is known about the protein residues involved in the oligomerization required for pore formation. Previous detailed analysis of the soluble three-dimensional structures of different wild-type and mutant actinoporins from Stychodactyla helianthus suggested residues which could be involved in this oligomerization. One of these stretches contains a conserved sequence compatible with an integrin-binding RGD motif. The results presented now deal with mutants affecting this motif in the well-characterized actinoporin sticholysin II. Small modifications along this three-residue sequence had profound effects on its solubility. Just a single methyl group yielded an RAD mutant version with a highly diminished haemolytic activity and altered oligomerization behaviour. The results obtained are discussed in terms of a key role for the RGD motif in maintaining the actinoporins' pore-competent state of protein oligomerization. Structured digital abstract and by (, ) [ABSTRACT FROM AUTHOR]

The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins. [ABSTRACT FROM AUTHOR]

Abstract: Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding specificity for this lipid. In this study, we have examined the role of the hydrogen bonding groups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylated SM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-affinity SM/StnII-interaction. [Copyright &y& Elsevier]

Abstract: Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins. [Copyright &y& Elsevier]

Abstract: Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen Hirsutella thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and α-sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells. [Copyright &y& Elsevier]

Within the last 10 years, the use of different RNases as therapeutic agents for various diseases has been pursued. Furthermore, the advancements of recombinant technology have allowed the assembly of proteins with different functions. In this regard, immunoribonucleases (immunoRNases) stand out as some of the most promising therapeutic candidates given their enzymatic and non-mutagenic character. Accordingly, the work reported here describes fusing RNase T1, one of the most studied members of the microbial RNase family, to the single-chain variable fragment (scFv) of a monoclonal antibody that targets the glycoprotein A33 antigen (GPA33) from human colon cancer cells. A heterologous production system, which employs the yeast Pichia pastoris, has been optimized to produce this immunoRNase (scFvA33T1) with yields of ∼ 5-10 mg·L−1. The purified protein appears to be correctly folded as it retains its antigen specificity and ribonucleolytic activity. Finally, it also shows specific binding to, internalization into and toxicity against GPA33-positive cell lines compared with the control, GPA33-negative cells. Overall, it can be concluded that scFvA33T1 is a promising therapeutic fusion protein with the additional advantage that presumably it can be produced and purified in large amounts using an easily scalable yeast-based system. [ABSTRACT FROM AUTHOR]

A single-chain fusion protein that directed the cytolytic activity of α-sarcin to A33 tumor antigen expressing cells was constructed and shown to effectively kill targeted cells. Glycoprotein A33 (GPA33) is a well-known colon cancer marker and a humanized antibody against it was used to target the α-sarcin. The fungal ribotoxin α-sarcin is one of the most potent and specific toxins known. It is small, protease resistant, thermostable and highly efficient towards the inactivation of ribosomes. This work describes the production and characterization of an immunotoxin resulting from fusing the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33 to fungal α-sarcin. This chimeric protein (scFvA33αsarcin), produced in Pichia pastoris and purified in high yield was proven to be properly folded, active, specific and stable. It showed high specific toxicity against GPA33-positive tumoral cell lines providing scientific evidence to sustain that scFvA33αsarcin is a good immunotherapeutic candidate against GPA33-positive colon carcinomas. [ABSTRACT FROM PUBLISHER]

The pathogenesis-related PR-1-like protein family comprises secreted proteins from the animal, plant, and fungal kingdoms whose biological function remains poorly understood. Here we have characterized a PR-1-like protein, Fpr1, from Fusarium oxysporum, an ubiquitous fungal pathogen that causes vascular wilt disease on a wide range of plant species and can produce life-threatening infections in immunocompromised humans. Fpr1 is secreted and proteolytically processed by the fungus. The fpr1 gene is required for virulence in a disseminated immunodepressed mouse model, and its function depends on the integrity of the proposed active site of PR-1-like proteins. Fpr1 belongs to a gene family that has expanded in plant pathogenic Sordariomycetes. These results suggest that secreted PR-1-like proteins play important roles in fungal pathogenicity. [ABSTRACT FROM AUTHOR]

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date. [ABSTRACT FROM AUTHOR]

Abstract: Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins. [Copyright &y& Elsevier]

Abstract: Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins. [Copyright &y& Elsevier]

Abstract: Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water–membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound. [Copyright &y& Elsevier]

Actinoporins are a family of sea anemone proteins that bind to membranes and produce functional pores which result in cell lysis. Actinoporin variants with decreased lytic activity usually show a reduced affinity for membranes. However, for some of these mutant versions there is no direct correlation between the loss of binding affinity and the decrease in their overall lytic activity, suggesting that other steps in pore formation may be hampered or facilitated by the mutations. To test this hypothesis on the mechanism of pore formation by this interesting family of proteins, structural and dynamic NMR studies have been carried out on two disabled variants of the actinoporin Sticholysin II, R29Q and Y111N. It is shown that their lytic activity is not only related to their membrane affinity but also to their conformational mechanism for membrane insertion. Alterations in their activities can be explained by structural, electrostatic and dynamic differences in a cluster of aromatic moieties and the N-terminus. In addition, the dynamic properties of some segments located at the C-terminus of the R29Q variant suggest a relevant role for this region in terms of protein-protein interactions. On the basis of all these results, we propose that R29 anchors a network of electrostatic interactions crucial for the actinoporin's approach to the membrane and that Y111 induces a necessary disorder in the loop regions that bind to membranes. Actinoporins are a family of sea anemone proteins that bind to membranes and produce functional pores. In this work, the structural and dynamic NMR data on two disabled variants of Sticholysin II, R29Q and Y111N, support the importance of a network of electrostatic interactions anchored by R29, and the local disorder at Y111, for actinoporin membrane binding and lysis. [ABSTRACT FROM AUTHOR]

Abstract: The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP∗) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP∗ produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches. [Copyright &y& Elsevier]

Abstract: Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1. Superposition of the three-dimensional structures of RNases T1 and U2 suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in Pichia pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast α-factor signal peptide. In addition, it has been shown that elimination of the Cys 1–Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein. [Copyright &y& Elsevier]

Antifungal protein (AFP) from Aspergillus giganteus was assayed for toxicity against the Fusarium oxysporum wild-type strain and mutants in genes involved in cell signaling (ΔpacC, pacCc Δfmk1) or cell-wall biogenesis (ΔchsV, Δchs7, Δgas1). The mutants were classified into two groups according to their sensitivity to AFP: ΔpacC, Δgas1 and Δchs7, which were significantly more resistant to AFP than the wild-type, and pacCC, Δfmk1 and ΔchsV, which were more sensitive. Western blot analysis revealed increased binding of AFP to the three resistant mutants, ΔpacC, Δgas1 and Δchs7, but also to ΔchsV, indicating that differential binding may not be a key determinant for sensitivity. Addition of Ca2+ or K+ dramatically reduced antifungal activity and binding of AFP, suggesting that these cations compete for the same targets as AFP at the surface of the fungal cell. [ABSTRACT FROM AUTHOR]

Abstract: The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid–protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7×108 M−1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid–water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation. [Copyright &y& Elsevier]

Abstract: Fungal ribotoxins are a family of extracellular ribonucleases which inhibit protein biosynthesis by inactivating the ribosomes. This inactivation results in the induction of cell death by apoptosis. Ribotoxins show antitumoral properties based on their ability to cross the membrane of some transformed cells. Unfortunately, they also show an unspecific cytotoxicity which has greatly impaired their potential clinical uses. α-Sarcin, produced by Aspergillus giganteus, is the best-characterized ribotoxin. Asp f 1, another ribotoxin produced by A. fumigatus, is indeed one of its major allergens. In this work, the Lactococcus lactis MG1363 strain has been engineered to produce and secrete not only wild-type Asp f 1 and α-sarcin but also three different mutants with reduced cytotoxicity and/or IgE-binding affinity. The proteins were secreted in native and active form when the extracellular medium employed was buffered at pH values around 8.0. Strains producing the wild-type natural α-sarcin were proved to be innocuous when administered intragastrically to mice for a period of 14 days. Overall, the results presented are discussed in terms of its potential application as a vehicle of oral delivery of hypoallergenic variants as well as a starting point to approach the design of strategies to accomplish the safe delivery of these proteins as antitumoral agents. [Copyright &y& Elsevier]

Abstract: The three-dimensional structures of the α-sarcin ribotoxin and its Δ(7–22) deletion mutant, both complexed with a 20-mer oligonucleotide mimicking the sarcin/ricin loop (SRL) of the ribosome, have been docked into the structure of the Halobacterium marismortui ribosome by fitting the nucleotide atomic coordinates into those of the ribosomal SRL. This study has revealed that two regions of the ribotoxin, residues 11–16 and 84–85, contact the ribosomal proteins L14 (residues 99–105) and L6 (residues 88–92), respectively. The first of these two ribotoxin regions appears to be crucial for its specific ribosome recognition. [Copyright &y& Elsevier]

15N–1H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin α-sarcin. The RDC values were obtained by partial alignment of α-sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69±0.12 Å for the backbone and 1.29±0.14 Å for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of α-sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure–function relationships concerning α-sarcin which can be reasonably extrapolated to other members of the ribotoxin family. [ABSTRACT FROM AUTHOR]

Asparagine 54 of a-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes. [ABSTRACT FROM AUTHOR]

α-Sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of α-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of α-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of α-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of α-sarcin and presumably of the other fungal ribotoxins. [ABSTRACT FROM AUTHOR]

Actinoporins constitute a family of α pore-forming toxins produced by sea anemones. The soluble fold of these proteins consists of a β-sandwich flanked by two α-helices. Actinoporins exert their activity by specifically recognizing sphingomyelin at their target membranes. Once there, they penetrate the membrane with their N-terminal α-helices, a process that leads to the formation of cation-selective pores. These pores kill the target cells by provoking an osmotic shock on them. In this review, we examine the role and relevance of the structural features of actinoporins, down to the residue level. We look at the specific amino acids that play significant roles in the function of actinoporins and their fold. Particular emphasis is given to those residues that display a high degree of conservation across the actinoporin sequences known to date. In light of the latest findings in the field, the membrane requirements for pore formation, the effect of lipid composition, and the process of pore formation are also discussed. • Actinoporins share a common soluble fold. • The actinoporin fold is exquisitely selected for sphingomyelin recognition. • Sticholysins' behavior depends on membrane composition and access to sphingomyelin. • Cholesterol greatly enhances sticholysin activity. [ABSTRACT FROM AUTHOR]

The ribosomal sarcin/ricin loop (SRL) is the target of ribosome-inactivating proteins like the N-glycosidase ricin and the fungal ribotoxin cr-sarcin. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL. Here we tested this hypothesis for the ribotoxin α-sarcin. Experiments with isolated ribosomes, cell-free translation systems, and viability assays with Saccharomyces cerevisiae strains defective in acidic stalk proteins showed that the inactivation exerted by α-sarcin is independent of the composition of the ribosomal stalk. Therefore, α-sarcin, with the same ribosomal target as ricin, seems to access the SRL by a different pathway. [ABSTRACT FROM AUTHOR]

Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades. However, regardless of the probe of choice, the results reported that StnII action was never able to empty the vesicles completely. This was hard to explain if StnII pores were to be stable and always leaky for the probes used. To address this question, we have used a variety of probes, including rhodamine 6G or Tb3+, to test the permeability of StnII's pores. Our results indicate that calcein was in fact too large to fit through StnII's pores, and that the standard method in the field is actually reporting StnII-induced transient permeation of the membrane rather than the passage of solutes through the stable assembled pores. In order to evaluate the permeability of these structures, we used a dithionite-based assay, which showed that the final pores were in fact open. Thus, our results indicate that the stable actinoporins' pores are open in spite of plateaued classic release curves. Besides the proper pore, the first stages of pore formation would inflict serious damage to living cells as well. Unlabelled Image • Negatively charged reporters are routinely used to study actinoporin's cation specific pores. • Data presented suggest that they only reflect transient instability of membrane impermeability. • Nevertheless, positively charged R6G seems to be a more sensitive probe for actinoporin's pores. • Size, rather than charge, is crucial for discriminating passage through actinoporins' pores. [ABSTRACT FROM AUTHOR]