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New Publication

Title   Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao
Abstract   Cystine knot toxins (CKTs) in spider venoms represent a rich source of novel ligands for varied ion channels. Here, we identified 95 novel
putative CKT precursors by analyzing expressed sequence tags of the tarantula Chilobrachys jingzhao venom gland. Phylogenetics analyses revealed one orphan family and six families with sequence similar-ity to known toxins. To further investigate the relationships of their structures, functions and evolu-tion, we assayed 10 representative toxins for their effect on ion channels, and performed structuremodel comparisons, evolution analysis and toxin distribution analysis.This study revealed twomajor types ofCKTs: pore-blocking toxins and gatingmodifier toxins.Afew blockers were observed with relatively high abun-dance and wide distribution, which may be a category of original toxins that block channels conserved in various preys with relatively high specificity. The gating modifier families contain advanced toxins, usually have many members and interact with diverse regulatory components of channels.
Keyword   Toxin, molecular diversity, classification, pore-blocking, gating modifier, evolution.
Author   J. Chen1, M. Deng1,Q.He1, E. Meng, L. Jiang, Z. Liao+, M. Rong and S. Liang*
New Publication

Title   A Novel Cell-Penetrating Peptide Sequence Derived by Structural Minimization of a Snake Toxin Exhibits Preferential Nucleolar Localization
Abstract   Structural simplification of a 42-residue venom peptideby N-to-C-terminal splicing led to two sequences [YKQCHKKGGXKKGSG, where X = nil (1) or 6-aminohexanoyl (2)], both efficiently uptaken by HeLa cells and, most interestingly, specifically localized at the nucleolus. Retro-2 was uptaken less efficiently, but a single (His → Ile) replacement recovered the translocation ability. None of the peptides were cytotoxic up to 100 μM. Enantio-1 did not translocate, suggesting that peptide uptake was receptor-mediated.
Keyword   Toxin, molecular diversity, classification, pore-blocking, gating modifier, evolution.
Author   Gandhi Rádis-Baptista, Beatriz G. de la Torre,† and David Andreu*†

Title   Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction
Abstract   The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36 h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3′-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.
Keyword   Fasciculin-2; AChE turnover; Synapse; Molecular chaperones; Protein folding; AChE assembly; RNA-binding protein; Translational regulation
Author   R.L. Rotundoa, b, C.A. Ruiza, E. Marreroa, L.M. Kimbella, S.G. Rossia, T. Rosenberryd, A. Darrb and P. Tsoulfasb

Title   Competitive Interactions of Collagen and a Jararhagin-derived Disintegrin Peptide with the Integrin α2-I Domain
Abstract   Integrin α2β1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the α2-I domain (α2-I), which binds fibrillar collagen via Mg2+-bridged interactions. The crystal structure of a truncated form of the α2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by α2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of α2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of α2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with 15N-labeled α2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of α2-I but does not induce a large scale conformational rearrangement of the I domain.
Author   Lester J. Lambert, Andrey A. Bobkov, Jeffrey W. Smith, and Francesca M. Marassi*
New Publication

Title   Electroencephalographic Evidence Of Brainstem Recruitment During Scorpion Envenomation
Abstract   Scorpion envenomation is a public health problem in Brazil, with most severe cases occuring in children under the age of 5 years (0.6% lethality). In fact, the toxic fractions of the Tityus serrulatus scorpion venom (TSSV) have greater permeability across the BBB of weanling rats when compared to adults. Although EEG alterations have been reported in up to 75% of pediatric severe cases, the role of the CNS in envenomation morbidity is still in debate. Our working hypothesis is that the neural substrates that play a major role in morbidity generate activity undetectable from EEG scalp leads. Twenty one-day-old rats(n=18) were injected s.c. with the deadliest toxic fraction of the TSSV, tityustoxin (TsTX; 2xDL50=6mg/kg). EEG leads were stereotaxicaly implanted in the Nucleus of the Solitary Tract (NTS) and left parietal cortex. EEG and ECG were continuously monitored by a video-EEG system until death or for a maximum period of 240 min. An experimental group pre-treated with carbamazepine (CBZ) was added in order to better access the cause-effect relationship between neural discharges and the systemic ECG alterations. High amplitude discharges in the NTS, which correlated to cardiac alterations, were recorded soon after administration of TsTX. Abnormal electrographic activity spread throughout the cortex only later in the recording. As expected, the CBZ treatment increased the latency for the first epileptiform discharge, decreased EEG/ECG alterations and increased the general survival time. In summary: peripheral scorpion toxin inoculation recruits brainstem involved in cardiovascular control and initial electrographic activity was undetectable from the cortical electrode.

Tityustoxin, Carbamazepine, Neuroprotection, Scorpion envenomation, Weanling rats.


Patrícia Alves Maia Guidine, Michel Bernanos Soares Mesquita, Tasso Moraes-Santos, André Ricardo Massensini, Márcio Flávio Dutra Moraes

Title   Structure-function relationship of bifunctional scorpion toxin BmBKTx1
Abstract   As the first identified scorpion toxin active on both big conductance Ca2+-activated K+ channels (BK) and small conductance Ca2+-activated K+ channels (SK), BmBKTx1 has been proposed to have two separate functional faces for two targets. To investigate this hypothesis, two double mutants, K21A-Y30A and R9A-K11A, together with wild-type toxin were expressed in Escherichia coli. The recombinant toxins were tested on cockroach BK and rat SK2 channel for functional assay. Mutant K21A-Y30A had a dramatic loss of function on BK but retained its function on SK. Mutant R9A-K11A did not lose function on BK or SK. These data support the two functional-face hypothesis and indicate that the BK face is on the C-terminal b-sheet.
Keyword   BmBKTx1; scorpion K+ channel toxin (KTx); K+ channel; two functional faces
Author   Suming Wang1,4, Lijun Huang2, Dieter Wicher3, Chengwu Chi2,4*, and Chenqi Xu*

Title   A common "hot spot" confers hERG blockade activity to α-scorpion toxins affecting K+ channels

While α-KTx peptides are generally known for their modulation of the Shaker-type and the Ca2+-activated potassium channels, γ-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily α-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative “hot spot” formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents. Biochem J 2004;378:745-52].In this work, we investigated if the “hot spot” is a commonality in subfamily α-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the “hot spot” in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active α-KTx. Additionally, we could extend our results to other α-KTx subfamily members belonging to α-KTx1, 4 and 6, therefore, the “hot spot” represents a common pharmacophore serving as a predictive tool for yet to be discovered α-KTxs.

Keyword   hERG channels; Scorpion toxins; K+ channels; α-KTx; γ-KTxs
Author   Yousra Abdel-Mottaleba, Gerardo Corzob, Marie-France Martin-Eauclairec, Honoo Sataked, Brigitte Céardc, Steve Peigneura, Praveen Nambarua, Pierre-Edouard Bougisc, Lourival D. Possanib and Jan Tytgata,
New Publication

Title   ArachnoServer: a database of protein toxins from spiders.
Abstract   Background: Venomous animals incapacitate their prey using complex venoms that can contain hundreds of unique protein toxins. The realisation that many of these toxins may have pharmaceutical and insecticidal potential due to their remarkable potency and selectivity against target receptors has led to an explosion in the number of new toxins being discovered and characterised. From an evolutionary perspective, spiders are the most successful venomous animals and they maintain by far the largest pool of toxic peptides. However, at present, there are no databases dedicated to spider toxins and hence it is difficult to realise their full potential as drugs, insecticides, and pharmacological probes.
Description: We have developed ArachnoServer, a manually curated database that provides detailed information about proteinaceous toxins from spiders. Key features of ArachnoServer include a new molecular target ontology designed especially for venom toxins, the most up-to-date taxonomic information available, and a powerful advanced search interface. Toxin information can be browsed through dynamic trees, and each toxin has a dedicated page summarising all available information about its sequence, structure, and biological activity. ArachnoServer currently manages 567 protein sequences, 334 nucleic acid sequences, and 51 protein structures.
Conclusions: ArachnoServer provides a single source of high-quality information about proteinaceous spider toxins that will be an invaluable resource for pharmacologists, neuroscientists, toxinologists, medicinal chemists, ion channel scientists, clinicians, and structural biologists. ArachnoServer is available online at www.arachnoserver.org.

Database, Spider toxin


Wood DL, Miljenovic T, Cai S, Raven RJ, Kaas Q, Escoubas P, Herzig V, Wilson D, King GF

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