Neuronal Prosurvival Role of Ceramide Synthase 2 by Olidogendrocyte-to-Neuron Extracellular Vesicle Transfer.

Ageing is associated with notorious alterations in neurons, i.e., in gene expression, mitochondrial function, membrane degradation or intercellular communication. However, neurons live for the entire lifespan of the individual. One of the reasons why neurons remain functional in elderly people is survival mechanisms prevail over death mechanisms. While many signals are either pro-survival or pro-death, others can play both roles. Extracellular vesicles (EVs) can signal both pro-toxicity and survival. We used young and old animals, primary neuronal and oligodendrocyte cultures and neuroblastoma and oligodendrocytic lines. We analysed our samples using a combination of proteomics and artificial neural networks, biochemistry and immunofluorescence approaches. We found an age-dependent increase in ceramide synthase 2 (CerS2) in cortical EVs, expressed by oligodendrocytes. In addition, we show that CerS2 is present in neurons via the uptake of oligodendrocyte-derived EVs. Finally, we show that age-associated inflammation and metabolic stress favour CerS2 expression and that oligodendrocyte-derived EVs loaded with CerS2 lead to the expression of the antiapoptotic factor Bcl2 in inflammatory conditions. Our study shows that intercellular communication is altered in the ageing brain, which favours neuronal survival through the transfer of oligodendrocyte-derived EVs containing CerS2.

t-boc synthesis of huwentoxin-i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy.

Tert-butyloxycarbonyl (t-Boc)-based native chemical ligation (NCL) techniques commonly employ hydrogen fluoride (HF) to create the thioester fragment required for the ligation process. Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin-I (HwTx-I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx-I has an X-Cys(16) -Cys(17) -X sequence mid-region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 µL with a 0.5-h incubation on ice, followed by 2.0 h at room temperature. RP-HPLC/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx-I N-terminal thioester fragment (Ala(1) -Cys(16) ), which was used in preparation for NCL. The resulting chemically ligated HwTx-I was oxidized/folded, purified, and then assessed for pharmacological target selectivity. Native-like HwTx-I produced by this method yielded an EC50 value of 340.5 ± 26.8 nM for Nav 1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav 1.3, this being similar to previous literature results using native material. This article represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 737-745, 2016.

Novel Conopeptides of Largely Unexplored Indo Pacific Conus sp.

Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage <50%). A third peptide, Asi3a from C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains.

Trancriptomic approach reveals the molecular diversity of Hottentotta conspersus (Buthidae) venom.

Scorpion venom consists of a complex mixture of molecules including biologically active compounds. Because of their high potency and selectivity, toxins have medical applicability. In the last decades, scorpion toxins have thus gained considerable interest among scientist in the fields of pharmacology, biophysics and neurobiology. Identification of scorpion venom peptides and toxins can be achieved based on transcriptome approaches. We constructed the first cDNA library and Expressed Sequence Tag (EST) study to explore the transcriptomic composition of the telson from the southern African scorpion Hottentotta conspersus, belonging to the family Buthidae. We obtained 21 new venom-related sequences (8 contigs and 16 singlets) from a total of 98 ESTs analyzed, including putative neurotoxins (chloride, potassium, sodium and calcium channel toxins), bradykinin-potentiating peptides and other venom peptides without established function. These novel toxin-related sequences might serve as basis for further research both of pharmaceutical and phylogenetic nature.

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms.

In the last decades, Ts1 has not only been the subject of many studies, it has also been considered as a very useful tool to investigate NaV channels and to explore the exact role of NaV channels in channelopathies. Ts1 is believed to modulate the activation process of NaV upon interaction at the neurotoxin binding site 4. Our aim was to carry out an in depth functional characterization of Ts1 on a wide array of Nav channels, in order to investigate its mechanism of action and to verify if Ts1 can indeed be considered as a prototype site 4 selective toxin, valid for all the Nav isoforms we know currently. Ts1 has been subjected to an in-depth functional investigation on 9 NaV isoforms expressed in Xenopus laevis oocytes. Ts1 does not only interfere with the activation process but also modulates the inactivation in a bell-shaped voltage-dependent matter. Furthermore, Ts1 altered the ion selectivity through insect NaV. without influencing the tetrodotoxin selectivity of the channels. Finally, Ts1 was also found to inhibit the sodium current through the cardiac Nav1.5 isoform. On the basis of the totally unexpected plethora of Nav modulations as induced by Ts1, we demonstrate that caution is required in interpretation the in vivo experiments when using Ts1. The electrophysiological characterization of Ts1 indeed shows that the general accepted contours of NaV binding sites are much more obscure than believed and that interpretation of NaV pharmacology upon toxin binding is more complex than believed thus far.

Partial transcriptomic profiling of toxins from the venom gland of the scorpion Parabuthus stridulus.

Since it is an apocrine secretion, scorpion venom is a complex mixture that contains a variety of low-molecular-weight basic proteins (neurotoxins), mucus, salts, as well as a large number of other constituents. Diversity of scorpion venom peptides exists also at the transcript level. Two kinds of venom peptides are typically considered: the neurotoxins and the antimicrobial peptides. We constructed a cDNA library and carried an EST (Expressed Sequence Tag) approach to overview the different peptides in the transcriptome of the telson from Parabuthus stridulus. P. stridulus are psammophilous and highly venomous scorpions endemic to Namibia (Prendini 2004) with medical relevance because of important human envenomation occurrence. We obtained 111 ESTs, 20% of them corresponding to cellular process transcripts, 7% to hypothetical proteins and 17% were sequences without good matches, but the majority of ESTs, 56%, corresponds to transcripts encoding for different venom components, including voltage-gated sodium, potassium and calcium channel toxins, antimicrobial peptides and other venom and cell proteins. To the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of the scorpion species P. stridulus, belonging to the family of medically important Buthidae scorpions. One hundred and eleven ESTs were analyzed, showing an important number of genes that encode for products similar to known scorpion venom components. In total, 17 unique and novel sequences were indentified. The identification and characterization of these compounds will be a good source of novel pharmacological tools for studying ion channels and the understanding of the physiological effects of toxins in P. stridulus envenomations at a molecular level.

Discovery of a new subclass of α-conotoxins in the venom of Conus australis.

Cone snails (Conus sp.) are poisonous animals that can be found in all oceans where they developed a venomous strategy to prey or to defend. The venom of these species contains an undeniable source of unique and potent pharmacologically active compounds. Their peptide compounds, called conotoxins, are not only interesting for the development of new pharmaceutical ligands, but they are also useful for studying their broad spectrum of targets. One conotoxin family in particular, the α-conotoxins, acts on nicotinic acetylcholine receptors (nAChRs) which dysfunctions play important roles in pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson's disease and Alzheimer's disease. Here we define a new subclass of the α-conotoxin family. We purified the venom of a yet unexplored cone snail species, i.e. Conus australis, and we isolated a 16-amino acid peptide named α-conotoxin AusIA. The peptide has the typical α-conotoxin CC-Xm-C-Xn-C framework, but both loops (m/n) contain 5 amino acids, which has never been described before. Using conventional electrophysiology we investigated the response of synthetically made globular (I-III, II-IV) and ribbon (I-IV, II-III) AusIA to different nicotinic acetylcholine receptors. The α7 nAChR was the only receptor found to be blocked with a similar potency by both peptide-configurations. This suggests that both α5/5 conotoxin isomers might be present in the venom gland of C. australis. NMR spectroscopy showed that no secondary structures define the peptides' three-dimensional topology. Moreover, the ribbon configuration, which is generally considered to be non-native, is more stable than the globular isomer. Accordingly, our findings show relevancy concerning the α-conotoxin classification which might be helpful in the design of novel therapeutic compounds.