Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous products from spider venoms, previously explored due to technical limitations. The chemical composition of spider venoms is diverse, ranging from low molecular weight organic compounds such as acylpolyamines to complex peptides. First, acylpolyamines (< 1000 Da) have an aromatic moiety linked to a hydrophilic lateral chain. They were characterized for the first time in spider venoms and are ligand-gated ion channel antagonists, which block mainly postsynaptic glutamate receptors in invertebrate and vertebrate nervous systems. Acylpolyamines represent the vast majority of organic components from the spider venom. Acylpolyamine analogues have proven to suppress hippocampal epileptic discharges. Moreover, acylpolyamines could suppress excitatory postsynaptic currents inducing Ca+ accumulation in neurons leading to protection against a brain ischemic insult. Second, short spider peptides (< 6000 Da) modulate ionic currents in Ca2+, Na+, or K+ voltage-gated ion channels. Such peptides may contain from three to four disulfide bridges. Some spider peptides act specifically to discriminate among Ca2+, Na+, or K+ ion channel subtypes. Their selective affinities for ion channel subfamilies are functional for mapping excitable cells. Furthermore, several of these peptides have proven to hyperpolarize peripheral neurons, which are associated with supplying sensation to the skin and skeletal muscles. Some spider N-type calcium ion channel blockers may be important for the treatment of chronic pain. A special group of spider peptides are the amphipathic and positively charged peptides. Their secondary structure is alpha-helical and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane. Acylpolyamines and peptides from spider venoms represent an interesting source of molecules for the design of novel pharmaceutical drugs.

No abstract

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and an FXa proteolytic cleavage region. This recombinant vector was transfected into E. coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/L of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9,392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9,400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7,538.6 Da as expected. Since native CssII is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na v 1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

SummaryThe profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-L-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N-and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.