The Androctonus australis garzoni scorpion venom contains toxins that selectively affect voltage-dependent K+-channels in cerebellum granular cells

A purified peptide from Androctonus australis Garzoni venom (AaG) affects selectively a K⁺-current recorded from cerebellum granular cells. This current is characterized by fast activating and inactivating kinetics similar to an I_A-type current. Addition of 2 μm peptide Aa1 (from Androctonus australis, toxin 1) to the external side of the channel suppressed completely and in a selective manner the I_A-type current, with an IC50 value of 130 nm, whereas in the same conditions, the other potassium current, identified as delayed rectifier (I_d), was not affected. Additionally, we show that another partially purified peptide (III-12) from the same venom was able to block reversibly both K⁺-currents. Received: 10 February 1997 / Accepted: 7 August 1997     

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.     

Evolutionary trace analysis of scorpion toxins specific for K-channels

Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.     

Gain-of-function screen for genes that affect Drosophila muscle pattern formation

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.     

Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels

1. We review the pharmacological actions of toxins present in the venom of the aggressive spider Phoneutria nigriventer.2. This venom is rich in toxins that affect ion channels and neurotransmitter release. Voltage-gated sodium, calcium, and potassium channels have been described as the main targets of these toxins.3. In addition to these classical actions Phoneutria toxins have also been shown to affect glutamate transporter.4. It is expected that molecular genetics in addition to biochemical, biophysical and pharmacological approaches will help to further define Phoneutria toxins and their mechanisms of action in the near future.     

A genome-wide screen for essential yeast genes that affect telomere length maintenance

Telomeres are structures composed of repetitive DNA and proteins that protect the chromosomal ends in eukaryotic cells from fusion or degradation, thus contributing to genomic stability. Although telomere length varies between species, in all organisms studied telomere length appears to be controlled by a dynamic equilibrium between elongating mechanisms (mainly addition of repeats by the enzyme telomerase) and nucleases that shorten the telomeric sequences. Two previous studies have analyzed a collection of yeast deletion strains (deleted for nonessential genes) and found over 270 genes that affect telomere length (Telomere Length Maintenance or TLM genes). Here we complete the list of TLM by analyzing a collection of strains carrying hypomorphic alleles of most essential genes (DAmP collection). We identify 87 essential genes that affect telomere length in yeast. These genes interact with the nonessential TLM genes in a significant manner, and provide new insights on the mechanisms involved in telomere length maintenance. The newly identified genes span a variety of cellular processes, including protein degradation, pre-mRNA splicing and DNA replication.     

Structural and functional diversity of acidic scorpion potassium channel toxins

Background

Although the basic scorpion K⁺ channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K⁺ channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly.

Principal Findings

Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif that has no apparent α-helixs and β-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K_d of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel.

Conclusions/Significance

These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.     

Molecular diversity and functional evolution of scorpion potassium channel toxins

Scorpion toxins affecting K(+) channels (KTxs) represent important pharmacological tools and potential drug candidates. Here, we report molecular characterization of seven new KTxs in the scorpion Mesobuthus eupeus by cDNA cloning combined with biochemical approaches. Comparative modeling supports that all these KTxs share a conserved cysteine-stabilized α-helix/β-sheet structural motif despite the differences in protein sequence and size. We investigated functional diversification of two orthologous α-KTxs (MeuTXKα1 from M. eupeus and BmP01 from Mesobuthus martensii) by comparing their K(+) channel-blocking activities. Pharmacologically, MeuTXKα1 selectively blocked Kv1.3 channel with nanomolar affinity (IC(50), 2.36 ± 0.9 nM), whereas only 35% of Kv1.1 currents were inhibited at 3 μM concentration, showing more than 1271-fold selectivity for Kv1.3 over Kv1.1. This peptide displayed a weak effect on Drosophila Shaker channel and no activity on Kv1.2, Kv1.4, Kv1.5, Kv1.6, and human ether-a-go-go-related gene (hERG) K(+) channels. Although BmB01 and MeuTXKα1 have a similar channel spectrum, their affinity and selectivity for these channels largely varies. In comparison with MeuTXKα1, BmP01 only exhibits a submicromolar affinity (IC(50), 133.72 ± 10.98 nM) for Kv1.3, showing 57-fold less activity than MeuTXKα1. Moreover, it lacks the ability to distinguish between Kv1.1 and Kv1.3. We also found that MeuTXKα1 inhibited the proliferation of activated T cells induced by phorbol myristate acetate and ionomycin at micromolar concentrations. Our results demonstrate that accelerated evolution drives affinity variations of orthologous α-KTxs on Kv channels and indicate that MeuTXKα1 is a promising candidate to develop an immune modulation agent for human autoimmune diseases.     

Unique interaction of scorpion toxins with the hERG channel

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.     

Chlorotoxin and related peptides: Short insect toxins from scorpion venom

Scorpion venom is a complex multicomponent mixture of biologically active substances, some of which possess very interesting properties and are used in quite unexpected areas. The family of chlorotoxin (CTX)-like peptides is a good example. These toxins exhibit insecticidal activity, however, the molecular mechanism of their action on the insect organism has not been established yet. Nevertheless, CTX-like peptides attracted considerable research effort due to their ability to interact specifically with cells of malignant brain tumors—gliomas. In the future these compounds may significantly simplify treatment of tumor diseases. The review summarizes the results accumulated over a forty years period of CTX-like peptides study. Aspects of their natural functions are considered, as well as the application area associated with gliomas.     

Refined solution structure of the anti-mammal and anti-insect LqqIII scorpion toxin: Comparison with other scorpion toxins

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatuswas refined and compared with other long-chain scorpion toxins. This structure, determined by ¹H-NMR and molecular modeling, involves an α-helix (18–29) linked to a three-stranded β-sheet (2–6, 33–39, and 43–51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 Å for Cα atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CSαβ (Cysteine Stabilized α-helix β-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na⁺ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins. Proteins 28:360–374, 1997 © 1997 Wiley-Liss, Inc.     

Drosophila genes that affect meiosis duration are among the meiosis related genes that are more often found duplicated

Using a phylogenetic approach, the examination of 33 meiosis/meiosis-related genes in 12 Drosophila species, revealed nine independent gene duplications, involving the genes cav, mre11, meiS332, polo and mtrm. Evidence is provided that at least eight out of the nine gene duplicates are functional. Therefore, the rate at which Drosophila meiosis/meiosis-related genes are duplicated and retained is estimated to be 0.0012 per gene per million years, a value that is similar to the average for all Drosophila genes. It should be noted that by using a phylogenetic approach the confounding effect of concerted evolution, that is known to lead to overestimation of the duplication and retention rate, is avoided. This is an important issue, since even in our moderate size sample, evidence for long-term concerted evolution (lasting for more than 30 million years) was found for the meiS332 gene pair in species of the Drosophila subgenus. Most striking, in contrast to theoretical expectations, is the finding that genes that encode proteins that must follow a close stoichiometric balance, such as polo, mtrm and meiS332 have been found duplicated. The duplicated genes may be examples of gene neofunctionalization. It is speculated that meiosis duration may be a trait that is under selection in Drosophila and that it has different optimal values in different species.     

Classification of scorpion toxins according to amino acid composition and sequence

Summary Scorpion venom toxins were systematically classified according to amino acid composition, insertion/deletion events and sequence. The significance of each comparison method and its outcome is discussed in relation to known immunological and structural properties. A general classification of the toxins is proposed that accounts for both the immunological groupings and the differences in mode of action.     

Development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies

Scorpion venoms contain toxic peptides that recognize K(+) channels of excitable and non-excitable cells. These toxins comprise three structurally distinct groups designated alpha-KTx, beta-KTx, and gamma-KTx. It is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. In this work, an expression vector (pTEV3) was constructed by inserting protein D (major capsid of phage lambda) and TEV protease recognition site into plasmid pET21d DNA sequences. Three alpha-KTx toxins (OsK2, PbTx1, and BmKK3) were cloned into vector pTEV3 and expressed as soluble fusion proteins. The fractions containing the purified fusion proteins (protein D-toxin) were treated with TEV protease to remove protein D. The resulting toxins were analyzed by MALDI-TOF Mass Spectrometry. The results showed that the vector is appropriate for the expression of the target toxins in soluble form and that ion exchange purification of these toxins by flow-through recovery is possible. Analysis by MALDI-TOF Mass Spectrometry of Osk2 demonstrated that this toxin was expressed in its native form, as suggested by the values expected for the presence of two disulfide bridges.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.