Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Modulation of insect Cav channels by peptidic spider toxins

Insects have a much smaller repertoire of voltage-gated calcium (Ca-v) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(v)1, Ca(v)2, and Ca(v)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca-v channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca-v channels, since severe loss-of-function mutations in genes encoding the pore-forming a, subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca-v channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca-v channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca-v channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca-v channels. This review focuses on peptidic spider toxins that specifically target insect Ca-v channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests. (c) 2006 Elsevier Ltd. All rights reserved.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Deduction of functional peptide motifs in scorpion toxins

Scorpion toxins are important physiological probes for characterizing ion channels. Molecular databases have limited functional annotation of scorpion toxins. Their function can be inferred by searching for conserved motifs in sequence signature databases that are derived statistically but are not necessarily biologically relevant. Mutation studies provide biological information on residues and positions important for structure-function relationship but are not normally used for extraction of binding motifs. 3D structure analyses also aid in the extraction of peptide motifs in which non-contiguous residues are clustered spatially. Here we present new, functionally relevant peptide motifs for ion channels, derived from the analyses of scorpion toxin native and mutant peptides. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.