Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 A and 18 A resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 A resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the beta sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.

Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics.

A potent hemolytic polypeptide, sticholysin II, has been purified to homogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is present in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, coencapsulated with its quencher N,N′-p-xylenebispyridinium bromide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing 20Ϫ25% cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being observed for 20Ϫ30% sphingomyelin molar content. The extent of the leakage is dependent on the molecular mass of the vesicle entrapped solutes in the range 445Ϫ960 Da. This suggests the involvement of a pore of about 1 nm in diameter based on the limiting size observed for the leakage of the different solutes. Oligomerization of the protein is apparently involved in the membrane permeabilization, based on the kinetic analysis of the leakage process which is shown to proceed through an all-or-none mechanism.Keywords : cytolysin; lipid vesicle; membrane permeabilization; protein-lipid interaction.Stichodactyla helianthus is a sea anemone occurring in the further isolated and resolved into two cytolytic proteins, sticholysin I and II, by ion-exchange chromatography (Díaz et al., coastal waters of the Caribbean region. Like other coelenterates, it produces many toxic peptides and proteins located within in-1992; Tejuca et al., 1996), that display very different primary structure. In contrast, it has been reported that two distinct anemtracellular specialized organelles called nematocysts and employed on the tentacles for defense and/or attack. These organ-one species produce a cytolytic protein of practically identical amino acid sequence. This is the case described for equinatoxin elles sting the prey by means of a rapid projection of a hollow tubule through which the toxic polypeptides are injected. The II from Actinia equina (Belmonte et al., 1994) and tenebrosin-C (Simpson et al., 1990) from Actinia tenebrosa. toxicity of these products, many of them are lethal substances, has been known for a long time. They can be divided into lowCytolysin III from S. helianthus, the most studied sea anemone toxin, was described as a potent hemolytic factor (Bernmolecular-mass (3Ϫ5 kDa) neurotoxins affecting sodium channels, and single polypeptide chain proteins (15Ϫ20 kDa), cyto-heimer and Avigad, 1976), although the extent of the effect was different depending on the origin of the mammalian erythrocytes lysins, acting on cell membranes (cytolytic effects towards red blood cells, platelets and fibroblasts have been reported ; Kem, considered. It was further demonstrated that the toxin increases the membrane permeability to small ions and solutes, probably 1988; Bernheimer...

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-Å resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a ␤-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the ␤-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the ⑀-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. ␣-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal ␤-hairpin in ribotoxins and to the different length of their unstructured loops. The aminoterminal deletion mutant ⌬(7-22) of ␣-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that ␣-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the ␤-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.