The diversity, origin, and evolution of chromoviruses in Eukaryota were examined using the massive amount of genome sequence data for different eukaryotic lineages. A surprisingly large number of novel full-length chromoviral elements were found, greatly exceeding the number of the known chromoviruses. These new elements are mostly structurally intact and highly conserved. Chromoviruses in the key Amniota lineage, the reptiles, have been analyzed by PCR to explain their evolutionary dynamics in amniotes. Phylogenetic analyses provide evidence for a novel centromere-specific chromoviral clade that is widespread and highly conserved in all seed plants. Chromoviral diversity in plants, fungi, and vertebrates, as shown by phylogenetic analyses, was found to be much greater than previously expected. The age of plant chromoviruses has been significantly extended by finding their representatives in the most basal plant lineages, the green and the red algae. The evolutionary origin of chromoviruses has been found to be no earlier than in Cercozoa. The evolutionary history and dynamics of chromoviruses can be explained simply by strict vertical transmission in plants, followed by more complex evolution in fungi and in Metazoa. The currently available data clearly show that chromoviruses indeed represent the oldest and the most widespread clade of Metaviridae.
Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13±20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105±120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.Keywords: acrylodan; biotin maleimide; cysteine-scanning mutagenesis; pore-forming protein; sea anemone.Pore-forming peptides and proteins are found in a variety of organisms, such as bacteria, plants, fungi, primitive metazoans, insects and humans [1±5]. They are mostly produced as watersoluble molecules destined to form pores in the lipid membranes of the host organism. Despite their diverse biological roles, most of them function by a common mode of action: they interact with cell or artificial lipid membranes, change conformation and oligomerize in the plane of the membrane to build water-filled pores that are permeable to solutes [6]. Insight into the assembly and operation of such pores calls for their three-dimensional structure. However, at present it is extremely hard to obtain the structure of protein complexes associated with lipids, as is true for membrane proteins [7]. So far, only the structure of a pore created by the bacterial cytolysin, Staphylococcus a-toxin Equinatoxin II (EqtII), a 179 amino-acid residue cytolysin from Actinia equina [18], belongs to the family of pore-forming proteins found in sea anemones [2]. Analysis o...
The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.
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