Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes

The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.     

Chemistry of some potent animal toxins

Conclusion I have discussed in this article only the most active toxins, with the result that many interesting substances have been omitted, e.g. the toxins from bee and wasp venoms (apamin, melittin, etc.), of many amphibians (bufotoxins, etc.), ciguatoxins, and many more. Poisons are found in every phylum except birds. Shrews exemplify venomous mammals. One gets a good illustration of the number of poisonous animals by studying the monumental and impressive work byHalstead ¹⁰⁶ which consequently excludes terrestrial animals. An interesting fact in this connection is that there are about 20,000 species of spiders, most of which are poisonous.A toxin ranking list has to be included in an article of this kind. The list is, of course, far from complete. Data on molecular weights, mouse lethal doses, etc. are lacking for many potent toxins, such as the dysentery toxin, a neurotoxin with a toxicity comparable to that of the botulinus toxins¹⁰⁷, the toxins from the jelly fishChironex fleckeri ¹⁰⁶.A comparison on molar basis gives a better notion of the toxicities. Curare has about 1/30 of the toxicity of the curarimimetic snake venom neurotoxins, clearly indicating that curare has a much lower affinity for the acetylcholine receptor.Toxic organisms have developed during millions of years more and more refined toxins, and this evolution has probably brought into existence toxins against every physiological function. Neurochemistry is to a great extent unexplored. Progress in this field will in the nearest future depend on specific toxins from various natural sources. Toxins from spiders, scorpions, snakes, frogs, and fishes are therefore not mere curiosities but valuable tools for research on the molecular mechanisms of neural function and synaptic transmission.     

Myotoxic phospholipases A from snake venom,Pseudechis colletti,producing myoglobinuria in mice

Summary 2 proteins producing myoglobinuria in mice were isolated from the venom of the Australian elapid snakePseudechis colletti and identified as phospholipases A showing close similarities in amino acid composition to a similarly acting enzyme from a sea snake venom (Enhydrina schistosa).     

麻痹性贝类毒素的安全评价与检测技术研究进展

麻痹性贝类毒素(Paralytic Shellfish Poisoning Toxins,PSTs)是一类高效的钠通道神经毒素,其作为赤潮毒索的一种主要由海洋中的部分双鞭毛藻产生,通过食物链进入人体产生毒性效应,是目前已知的影响范围最广、毒性最强、危害最大的一类海洋生物毒素,因而格外引发了人们对于此类毒素的关注。本文对PSTs的来源、传递方式、理化性质、安全性评价、检测技术等热点问题及发展趋势做了详细的介绍与展望。     

Modulation of phospholipase A2 activity generated by molecular evolution

Snake venom oligomeric neurotoxins offer several unique examples of modulation of phospholipase A2 (PLA2) activity generated by molecular evolution. This phenomenon was found in evolutionary younger snakes and is probably common for representatives of the genus Vipera. At present, the best-studied example is the heterodimeric neurotoxin vipoxin from the venom of the southeast European snake Vipera ammodytes meridionalis. It is a complex between a basic strongly toxic PLA2 and an acidic and catalytically inactive PLA2-like component (Inh). This is the first reported example of a high degree of structural homology (62%) between an enzyme and its natural protein inhibitor. The inhibitor is a product of the divergent evolution of the unstable PLA2 in order to stabilize it and to preserve the pharmacological activity/toxicity for a long time. Inh reduces both the catalytic activity and toxicity of PLA2. Vipoxin also illustrates evolution of the catalytic into a inhibitory function. Vipoxin analogues have been found in the venom of viperid snakes inhabiting diverse regions of the world. An attempt is made to explain modulation of the toxic function by the three-dimensional structure of vipoxin.     

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca²⁺ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca²⁺], derived from intracellular stores, followed, only in myotubes, by a large Ca²⁺ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca²⁺ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca²⁺ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death. Received 21 January 2009; received after revision 05 March 2009; accepted 11 March 2009     

Regulation of nicotinic acetylcholine receptors by tyrosine kinases in the peripheral and central nervous system: same players, different roles

Nicotinic acetylcholine receptors (nAChRs) exist in many subtypes and are found in the peripheral and central nervous system where they mediate or modulate synaptic transmission. We review how tyrosine phosphorylation and kinases regulate muscle and neuronal nAChRs. Interestingly, although some of the same kinase players interact with the various receptor subtypes, the functional consequences are different. While concerted action of MuSK, Abl- and Src-family kinases (SFKs) regulates the synaptic distribution of nAChRs at the neuromuscular junction, SFKs activate heteromeric neuronal nAChRs in adrenal chromaffin cells, thereby enhancing catecholamine secretion. In contrast, the activity of homomeric neuronal nAChRs, as found in the hippocampus, is negatively regulated by tyrosine phosphorylation and SFKs. It appears that tyrosine kinases provide the means to regulate all nAChRs; but the functional consequences, even those caused by the same kinase family, are specific for each receptor subtype and location. Received 21 February 2006; received after revision 24 July 2006; accepted 30 August 2006     

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

A large variety of snake toxins evolved from PLA₂ digestive enzymes through a process of ‘accelerated evolution’. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA₂s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca²⁺ concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells. Received 05 March 2008; received after revision 08 April 2008; accepted 29 April 2008     

Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α₁–α₂–α₃ non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α₁–α₃ structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α₁–α₃-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.     

Clostridial neurotoxins: structure-function led design of new therapeutics

The neurotoxins produced by various species of Clostridia are the causative agents of botulism and tetanus. The ability of the toxins, specifically those of the botulinum neurotoxin family, to disrupt neurotransmission has been exploited for use in several medical indications and now represents the therapeutic option of choice in a number of cases. Clostridial neurotoxins have been discovered to have a multi-domain structure that is shared between the various proteins of the family, and it has also been determined that each domain contributes a specific role to the holotoxin. The extensive use of recombinant expression approaches, along with solution of multiple crystallographic structures of individual domains, has enabled researchers to explore structurefunction relationships of the toxin domains more closely. These advances have facilitated a greater understanding of the potential use of individual domains for a wide variety of purposes, including the development of new therapeutics. Received 21 October 2005; received after revision 10 November 2005; accepted 16 November 2005     

Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.     

Erratum to: Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.     

Alternative exon usage selectively determines both tissue distribution and subcellular localization of the acyl-CoA thioesterase 7 gene products

Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A. Recent studies have demonstrated that one gene named Acot7, reported to be mainly expressed in brain and testis, is transcribed in several different isoforms by alternative usage of first exons. Strongly decreased levels of ACOT7 activity and protein in both mitochondria and cytosol was reported in patients diagnosed with fatty acid oxidation defects, linking ACOT7 function to regulation of fatty acid oxidation in other tissues. In this study, we have identified five possible first exons in mouse Acot7 (Acot7a–e) and show that all five first exons are transcribed in a tissue-specific manner. Taken together, these data show that the Acot7 gene is expressed as multiple isoforms in a tissue-specific manner, and that expression in tissues other than brain and testis is likely to play important roles in fatty acid metabolism. Received 5 February 2007: received after revision 3 April 2007; accepted 19 April 2007     

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steadystate length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells.     

NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS

NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns.     

Bacterial protein toxins and cell vesicle trafficking

A group of bacterial protein toxins interfere with vesicular trafficking inside cells. Clostridial neurotoxins affect mainly the highly regulated fusion of neurotransmitter- and hormone-containing vesicles with the plasma membrane. They cleave the three SNARE proteins: VAMP, SNAP-25 and syntaxin, and this selective proteolysis results in a blockade of exocytosis. TheHelicobacter pylori cytotoxin is implicated in the pathogenesis of gastroduodenal ulcers. It causes a progressive and extensive vacuolation of cells followed by necrosis, after a cytotoxin-induced alteration of membrane trafficking by late endosomes. Vacuoles originate from this compartment in a rab7-dependent process and swell because they are acidic and accumulate membrane-permeant amines.     

Isolation and characterization of hainantoxin-IV,a novel antagonist of tetrodotoxin-sensitive sodium channels from the Chinese bird spider <Emphasis Type="Italic">Selenocosmia hainana</Emphasis>

A neurotoxin, named hainantoxin-IV, was purified from the venom of the spider Selenocosmia hainana. The amino acid sequence was determined by Edman degradation, revealing it to be a 35-residue polypeptide amidated at its C terminal and including three disulfide bridges: Cys2-Cys17, Cys9-Cys24, and Cys16-Cys31 assigned by partial reduction and sequence analysis. Hainantoxin-IV shares 80% sequence identity with huwentoxin-IV from the spider S. huwena, a potent antagonist that acts at site 1 on tetrodotoxin-sensitive (TTX-S) sodium channels, suggesting that hainantoxin-IV adopts an inhibitor cystine knot structural motif like huwentoin-IV. Under whole-cell voltage-clamp conditions, this toxin has no effect on tetrodotoxin-resistant voltage-gated sodium channels in adult rat dorsal root ganglion neurons, while it blocks TTX-S sodium channels in a manner similar to huwentoxin-IV, and the actions of both toxins on sodium currents are very similar to that of tetrodotoxin. Thus, they define a new family of spider toxins affecting sodium channels.     

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ₂) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.