Disintegrin-like Protein Strategy to Inhibit Aggressive Triple-Negative Breast Cancer.

Venoms are a rich source of bioactive compounds, and among them is leberagin-C (Leb-C), a disintegrin-like protein derived from the venom of Macrovipera lebetina transmediterrannea snakes. Leb-C has shown promising inhibitory effects on platelet aggregation. Previous studies have demonstrated that this SECD protein specifically targets α5ß1, αvß3, and αvß6 integrins through a mimic mechanism of RGD disintegrins. In our current study, we focused on exploring the potential effects of Leb-C on metastatic breast cancer. Our findings revealed that Leb-C disrupted the adhesion, migration, and invasion capabilities of MDA-MB-231 breast cancer cells and its highly metastatic D3H2LN sub-population. Additionally, we observed significant suppression of adhesion, migration, and invasion of human umbilical vein endothelial cells (HUVECs). Furthermore, Leb-C demonstrated a strong inhibitory effect on fibroblast-growth-factor-2-induced proliferation of HUVEC. We conducted in vivo experiments using nude mice and found that treatment with 2 µM of Leb-C resulted in a remarkable 73% reduction in D3H2LN xenograft tumor size. Additionally, quantification of intratumor microvessels revealed a 50% reduction in tumor angiogenesis in xenograft after 21 days of twice-weekly treatment with 2 µM of Leb-C. Collectively, these findings suggest the potential utility of this disintegrin-like protein for inhibiting aggressive and resistant metastatic breast cancer.

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis.

Development and homeostasis of the vascular system requires integrin-promoting endothelial cell adhesion, migration and survival. Nowadays, integrins represent potential targets for pharmacological agents and open new avenues for the control of metastatic spread in the treatment of tumor malignancies. We have already reported that PIVL, a serine protease inhibitor isolated from Macrovipera lebetina venom, displays an anti-tumor effect through interference with integrin receptor function. Here, we report that PIVL inhibits human vascular endothelial cell adhesion and migration onto fibrinogen and fibronectin in a dose-dependent manner without any cytotoxicity. Furthermore, we show that PIVL increases microtubule dynamic instability in HMEC-1 transfected with EGFP-tagged α-tubulin. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrate that PIVL exhibits a strong anti-angiogenic effect both in vitro and in vivo. Interestingly, results herein reveal that the potent anti-angiogenic properties of PIVL are mediated by its RGD-like motif ((41)RGN(43)).

Subtype-selective activation of K(v)7 channels by AaTXKß2₋64, a novel toxin variant from the Androctonus australis scorpion venom.

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKß2₋64, a novel variant of AaTXKß1₋64, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKß2₋64, but not AaTXKß1₋64, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKß2₋64 also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKß2₋64-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.

Pre-clinical studies of toxin-specific nanobodies: evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming.

Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab'(2) based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of (99m)Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab'(2) product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab'(2) based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10.

Neutralizing Dromedary-Derived Nanobodies Against BotI-Like Toxin From the Most Hazardous Scorpion Venom in the Middle East and North Africa Region.

Scorpion envenoming is a severe health problem in many regions causing significant clinical toxic effects and fatalities. In the Middle East/North Africa (MENA) region, Buthidae scorpion stings are responsible for devastating toxic outcomes in human. The only available specific immunotherapeutic treatment is based on IgG fragments of animal origin. To overcome the limitations of classical immunotherapy, we have demonstrated the in vivo efficacy of NbF12-10 bispecific nanobody at preclinical level. Nanobodies were developed against BotI analogues belonging to a distinct structural and antigenic group of scorpion toxins, occurring in the MENA region. From Buthus occitanus tunetanus venom, BotI-like toxin was purified. The 41 N-terminal amino acid residues were sequenced, and the LD50 was estimated at 40 ng/mouse. The BotI-like toxin was used for dromedary immunization. An immune VHH library was constructed, and after screening, two nanobodies were selected with nanomolar and sub-nanomolar affinity and recognizing an overlapping epitope. NbBotI-01 was able to neutralize 50% of the lethal effect of 13 LD50 BotI-like toxins in mice when injected by i.c.v route, whereas NbBotI-17 neutralized 50% of the lethal effect of 7 LD50. Interestingly, NbBotI-01 completely reduced the lethal effect of the 2 LD50 of BotG50 when injected at 1:4 molar ratio excess. More interestingly, an equimolar mixture of NbBotI-01 with NbF12-10 neutralized completely the lethal effect of 7 and 5 LD50 of BotG50 or AahG50, at 1:4 and 1:2 molar ratio, respectively. Hence, NbBotI-01 and NbF12-10 display synergic effects, leading to a novel therapeutic candidate for treating Buthus occitanus scorpion stings in the MENA region.

A bispecific nanobody to provide full protection against lethal scorpion envenoming.

Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.

CC-PLA2-1 and CC-PLA2-2, two Cerastes cerastes venom-derived phospholipases A2, inhibit angiogenesis both in vitro and in vivo.

Integrins are essential in the complex multistep process of angiogenesis and are thus attractive targets for the development of antiangiogenic therapies. Integrins are antagonized by disintegrins and C-type lectin-like proteins, two protein families from snake venom. Here, we report that CC-PLA2-1 and CC-PLA2-2, two novel secreted phospholipases A(2) (PLA(2)) isolated from Cerastes cerastes venom, also showed anti-integrin activity. Indeed, both PLA(2)s efficiently inhibited human brain microvascular endothelial cell adhesion and migration to fibrinogen and fibronectin in a dose-dependent manner. Interestingly, we show that this anti-adhesive effect was mediated by alpha5beta1 and alphav-containing integrins. CC-PLA2s also impaired in vitro human brain microvascular endothelial cell tubulogenesis on Matrigel and showed antiangiogenic activity in vivo in chicken chorioallantoic membrane assay. The complete PLA(2) cDNAs were cloned from a venom gland cDNA library. Mature CC-PLA2-1 and CC-PLA2-2 contain 121 and 120 amino acids, respectively, including 14 cysteines each and showed 83% identity. Tertiary model structures of CC-PLA2-1 and CC-PLA2-2 were generated by homology modeling. This is thus the first study describing an antiangiogenic effect for snake venom PLA(2)s and reporting first clues to their mechanism of action on endothelial cells.

Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venom.

Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.

C-type lectin protein isoforms of Macrovipera lebetina: cDNA cloning and genetic diversity.

Snake venom contains a complex protein mixture belonging to a few well-characterized protein families: disintegrins, phospholipase A2, serine protease, l-amino acid oxidase, Zn-dependent metalloproteinase, natriuretic peptides, myotoxins, cysteine-rich secretory protein (CRISP) toxins, Kunitz-type protease inhibitors and C-type lectin-like. Despite their pharmacological importance, little is known about the exact composition of each protein family. We report here the cloning of 25 complete ORFs from Macrovipera lebetina transmediterranea venom gland that encodes several isoforms and novel C-type lectins (CTLs). 16 alpha and nine beta CTL chains were identified. Based on their sequence alignment, we categorized the 16 CTL alpha subunits into five groups and the nine CTL beta subunits into four groups to deduce the phylogenetic tree of M. lebetina transmediterranea CTLs. Sequence analysis revealed that they share a high degree of similarity with each other and with other snake venom CTLs. The M. lebetina transmediterranea CTL sequences described here contain a C-lectin carbohydrate recognition domain-like fold (C-lectin CRD-like) characterized by several conserved amino acid residues in their structure, especially the cysteine. Finally, based on the comparison of some Macrovipera CTL, we propose that some new CTL gene versions should have occurred through "domains shuffling" from former genes.

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins.

BACKGROUND: K+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels. RESULTS: Four multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins.Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins.Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors.Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences. CONCLUSION: The phylogenetic results were in perfect agreement with those obtained by the CART algorithm.Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.

Hemitoxin, the first potassium channel toxin from the venom of the Iranian scorpion Hemiscorpius lepturus.

Hemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity.

Immunological characterization of a non-toxic peptide conferring protection against the toxic fraction (AahG50) of the Androctonus australis hector venom.

KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.

Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels.

In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel.

Targeting α1 inserted domain (I) of α1ß1 integrin by Lebetin 2 from M. lebetina transmediterranea venom decreased tumorigenesis and angiogenesis.

Through the recent development of knowledge in biotechnology and bioinformatics, snake venoms are widely used to develop new drugs to treat diseases such as hypertension and cancer. We have previously reported that Lebetin 2 isolated from Macrovipera lebetina transmediterranea venom displays a potent anti-platelet activity and exerts a cardioprotective effect in ischemia-reperfusion (IR) injury model. Here, we report that Lebetin 2 possess an anti-tumor effect by targeting the integrin receptor function. It was thus able to inhibit both adhesion and migration of pheochromocytoma cells (PC12) and α1ß1 integrin-expressing CHO cells (CHO-α1) to type I and IV collagens. Moreover, this peptide affects proliferation of PC12 cells by modulating AKT phosphorylation. Furthermore, Lebetin 2 exhibits a potent anti-angiogenic effect as assessed in vitro and ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Interestingly, the interaction mode of Lebetin 2 with the integrin α1ß1, assessed in silico, showed that the peptide represents a steric obstruction preventing the collagen from enforcing the interactions with the integrin.

Lipid Biomarkers in Alzheimer's Disease.

BACKGROUND: There are now significant evidences that lipid metabolism is affected in numerous neurodegenerative diseases including Alzheimer's disease. These dysfunctions lead to abnormal levels of certain lipids in the brain, cerebrospinal fluid and plasma. It is consequently of interest to establish lipid profiles in neurodegenerative diseases. This approach, which can contribute to identify lipid biomarkers of Alzheimers' disease, can also permit to identify new therapeutic targets. It was therefore of interest to focus on central and peripheral biomarkers in Alzheimer's disease. METHODS: A review of the literature on 148 papers was conducted. Based on this literature, the involvement of lipids (cholesterol and oxysterols, fatty acids, phospholipids) in Alzheimer's disease has been proposed. RESULTS: Of the 148 references cited for lipid biomarkers for Alzheimer's disease, 65 refer to cholesterol and oxysterols, 35 to fatty acids and 40 to phospholipids. Among these lipids, some of them such as 24S-hydroxyckolesterol, open up new therapeutic perspectives in gene therapy, in particular. The results on the very long-chain fatty acids suggest the potential of peroxisomal dysfunctions in Alzheimer's disease. As for the phospholipids, they could constitute interesting biomarkers for detecting the disease at the prodromal stage. CONCLUSION: There are now several lines of evidence that lipids play fundamental roles in the pathogenesis of AD and that some of them have a prognostic and diagnosis value. This may pave the way for the identification of new therapeutic targets, new effective drugs and / or new treatments.

Modulation of Kv3.1b potassium channel level and intracellular potassium concentration in 158N murine oligodendrocytes and BV-2 murine microglial cells treated with 7-ketocholesterol, 24S-hydroxycholesterol or tetracosanoic acid (C24:0).

Little is known about K+ regulation playing major roles in the propagation of nerve impulses, as well as in apoptosis and inflammasome activation involved in neurodegeneration. As increased levels of 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC) and tetracosanoic acid (C24:0) have been observed in patients with neurodegenerative diseases, we studied the effect of 24 and/or 48 h of treatment with 7KC, 24S-OHC and C24:0 on Kv3.1b potassium channel level, intracellular K+ concentration, oxidative stress, mitochondrial dysfunction, and plasma membrane permeability in 158N oligodendrocytes and BV-2 microglial cells. In 158N cells, whereas increased level of Kv3.1b was only observed with 7KC and 24S-OHC but not with C24:0 at 24 h, an intracellular accumulation of K+ was always detected. In BV-2 cells treated with 7KC, 24S-OHC and C24:0, Kv3.1b level was only increased at 48 h; intracellular K+ accumulation was found at 24 h with 7KC, 24S-OHC and C24:0, and only with C24:0 at 48 h. Positive correlations between Kv3.1b level and intracellular K+ concentration were observed in 158N cells in the presence of 7KC and 24S-OHC, and in 7KC-treated BV-2 cells at 48 h. Positive correlations were also found between Kv3.1b or the intracellular K+ concentration, overproduction of reactive oxygen species, loss of transmembrane mitochondrial potential and increased plasma membrane permeability in 158N and BV-2 cells. Our data support that the lipid environment affects Kv3.1b channel expression and/or functionality, and that the subsequent rupture of K+ homeostasis is relied with oligodendrocytes and microglial cells damages.

Lebectin and lebecetin, two C-type lectins from snake venom, inhibit alpha5beta1 and alphaV-containing integrins.

Integrins are essential protagonists in the complex multistep process of cancer progression and metastasis. We recently reported that lebectin, a novel C-type lectin from Macrovipera lebetina venom, displays an anti-integrin activity. In this study, we extend this observation to lebecetin, a second C-type lectin isolated from the same venom and previously reported as a potent inhibitor of platelet aggregation. Both venom lectins appear to exert their effect on cell adhesion, migration, invasion and proliferation by inhibiting alpha5beta1 and alphav-containing integrins. Moreover, the inhibition of alpha5beta1 and alphav integrins is likely due to the binding of venom peptides, as both lebectin and lebecetin co-immunoprecipitate with these integrins. Lebectin and lebecetin are thus the first examples of venom C-type lectins inhibiting an integrin other than the collagen receptor alpha2beta1.

A novel depolarizing activity of scorpion venom fraction M1 due to activation of skeletal muscle nicotinic receptors.

A depolarizing activity following interaction with nicotinic acetylcholine receptors (nAchRs) in skeletal muscle cells, was observed for the first time in the non-toxic venom fraction (M1) of the yellow scorpion Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by recording changes in [Ca2+]i. When applied, M1 (10 microg/mL) induced a transient increase of [Ca2+]i which could be blocked by a prior application of alpha-Bungarotoxin (alpha-Bg-Tx).

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system.

We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.

Evidence of K+ homeostasis disruption in cellular dysfunction triggered by 7-ketocholesterol, 24S-hydroxycholesterol, and tetracosanoic acid (C24:0) in 158N murine oligodendrocytes.

Imbalance in the homeostasis of K+ ions has been reported to contribute to the pathogenesis of neurodegenerative diseases. 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC), and tetracosanoic acid (C24:0), often found at increased levels in patients with Alzheimer's disease, Multiple Sclerosis and X-ALD, are able to trigger numerous nerve cell dysfunctions. We therefore studied the impact of 7KC, 24S-OHC, and C24:0 on 158N murine oligodendrocytes, and determined their impact on K+ homeostasis. The effects of 7KC, 24S-OHC and C24:0 on lipid membrane organization and membrane potential were examined with merocyanine 540 (MC540) and bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3)), respectively. The intracellular concentration of K+ ([K+]i) was measured by flame photometry and the ratiometric approach using the PBFI-AM fluorescence indicator. To determine the relationships between [K+]i and lipotoxicity, 158N cells were pre-treated with a universal Kv channels blocker, 4-aminopyridine (4-AP), without or with 7KC, 24S-OHC or C24:0. Cell adhesion, cell growth, mitochondrial depolarization, cytoplasmic membrane integrity, the presence of SubG1 and the morphological aspect of the nuclei were determined with various microscopy, flow cytometry and biochemistry methods. 7KC, 24S-OHC and C24:0 induced changes in lipid content and polarization of the cytoplasmic membrane. These events were associated with increased [K+]i. Blocking Kv channels with 4-AP exacerbated 7KC-, 24S-OHC- and C24:0-induced cell dysfunction. 4-AP exacerbated loss of cell adhesion and cell growth inhibition, amplified mitochondrial depolarization and cytoplasmic membrane damage, and increased the percentage of SubG1 cells. The positive correlation between [K+]i and cell death supports the potential involvement of K+ in 7KC-, 24S-OHC-, and C24:0-induced cytotoxicity.