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.

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.

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.

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.

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.

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.

The small subunit of Hemilipin2, a new heterodimeric phospholipase A2 from Hemiscorpius lepturus scorpion venom, mediates the antiangiogenic effect of the whole protein.

In a previous study, we reported the identification of Hemilipin, the first secreted heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom and demonstrated its effective inhibition of all angiogenesis key steps in vitro and in vivo. Here, we aimed to characterize a second sPLA2, Hemilipin2, from the same venom and to elucidate its antiangiogenic effect. The protein was purified by chromatography separation and analyzed by MALDI/TOF mass spectrometry. Its N terminal amino acid sequence was determined by Edman degradation method and the enzymatic activity by fatty acids release assay. Hemilipin2 antiangiogenic activity was investigated by studying its effect in vitro on adhesion, migration and capillary like tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Artery Endothelial Cells (HPAECs); and in vivo on the chick embryo chorioallantoic membrane (CAM) assay. Data to be presented show that Hemilipin2 is heterodimeric composed by two subunits: the large one has a molecular weight of 12,866 and the small one of 2461 a.m.u. It has a strong calcium-dependent PLA2 activity and impacts angiogenesis in vitro and in vivo without showing any cytotoxic or apoptotic signs. Its chemical modification with p-Bromophenacyl Bromide abolishes the enzymatic activity without affecting the antiangiogenic effect. Furthermore, it has been proved that Hemilipin2 small subunit was able to inhibit blood vessel formation both in vitro and in vivo. These findings may serve as a starting point for the designing of a new generation of specific inhibitor of human angiogenesis at different steps.

Dromedary immune response and specific Kv2.1 antibody generation using a specific immunization approach.

Voltage-gated potassium (Kv) channels form cells repolarizing power and are commonly expressed in excitable cells. In non-excitable cells, Kv channels such as Kv2.1 are involved in cell differentiation and growth. Due to the involvement of Kv2.1 in several physiological processes, these channels are promising therapeutic targets. To develop Kv2.1 specific antibody-based channel modulators, we applied a novel approach and immunized a dromedary with heterologous Ltk- cells that overexpress the mouse Kv2.1 channel instead of immunizing with channel protein fragments. The advantage of this approach is that the channel is presented in its native tetrameric configuration. Using a Cell-ELISA, we demonstrated the ability of the immune serum to detect Kv2.1 channels on the surface of cells that express the channel. Then, using a Patch Clamp electrophysiology assay we explored the capability of the dromedary serum in modulating Kv2.1 currents. Cells that were incubated for 3h with serum taken at Day 51 from the start of the immunization displayed a statistically significant 2-fold reduction in current density compared to control conditions as well as cells incubated with serum from Day 0. Here we show that an immunization approach with cells overexpressing the Kv2.1 channel yields immune serum with Kv2.1 specific antibodies.

Biochemical and monolayer characterization of Tunisian snake venom phospholipases.

The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers'venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases.

Interaction of a snake venom L-amino acid oxidase with different cell types membrane.

Snake venom l-amino acid oxidases are multifunctional enzymes that exhibited a wide range of pharmacological activities. Although it has been established that these activities are primarily caused by the H2O2 generated in the enzymatic reaction, the molecular mechanism, however, has not been fully investigated. In this work, LAAO interaction with cytoplasmic membranes using different cell types and Langmuir interfacial monolayers was evaluated. The Cerastes cerastes venom LAAO (CC-LAAO) did not exhibit cytotoxic activities against erythrocytes and peripheral blood mononuclear cells (PBMC). However, CC-LAAO caused cytotoxicity on several cancer cell lines and induced platelet aggregation in dose-dependent manner. Furthermore, the enzyme showed remarkable effect against Gram-positive and Gram-negative bacteria. These activities were inhibited on the addition of catalase or substrate analogs, suggesting that H2O2 liberation× is required for these effects. Binding studies revealed that CC-LAAO binds to the cell surface and enables the production of highly localized concentration of H2O2 in or near the binding interfaces. On another hand, the interaction of CC-LAAO with a mimetic phospholipid film was evaluated, for the first time, using a monomolecular film technique. Results indicated that phospholipid/CC-LAAO interactions are not involved in their binding to membrane and in their pharmacological activities.

Hemilipin, a novel Hemiscorpius lepturus venom heterodimeric phospholipase A2, which inhibits angiogenesis in vitro and in vivo.

Phospholipases A2 (PLA2) are enzymes which specifically hydrolyze the sn-2 acyl ester bond of phospholipids producing free fatty acids and lysophospholipids. The secreted PLA2 (sPLA2) are the most common types of PLA2 purified from the snake venom, mammalian pancreatic juice and other sources. They display a variety of toxic actions and biological activities, including antitumoral and antiangiogenic effects. In this study, we report the isolation, characterization and the antiangiogenic activity of Hemilipin, a novel sPLA2 extracted from Hemiscorpius lepturus venom, the most dangerous scorpion in Iran. Hemilipin was purified by HPLC and analyzed by MALDI TOF/MS. The primary structure was determined by EDMAN degradation method and the PLA2 activity by titration of fatty acids released from the egg yolk phospholipids. Its antiangiogenic activity was studied in vitro by evaluating effects on apoptosis, Matrigel angiogenesis, migration and adhesion of human umbilical vein endothelial cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) and in vivo by the chorioallantoic membrane (CAM) assay. Mass spectrometry profile showed that Hemilipin is heterodimeric and the PLA2 test demonstrated its strong hydrolytic activity. N-terminal aminoacid sequence highlighted a significant homology of Hemilipin's small and large subunits with other sPLA2 group III. Hemilipin had no effect on apoptosis, but strongly impacted angiogenesis both in vitro and in vivo. Our results demonstrate that this novel non toxic sPLA2 could be a new tool to disrupt at different steps human angiogenesis.

AaTX1, from Androctonus australis scorpion venom: purification, synthesis and characterization in dopaminergic neurons.

We have purified the AaTX1 peptide from the Androctonus australis (Aa) scorpion venom, previously cloned and sequenced by Legros and collaborators in a venom gland cDNA library from Aa scorpion. AaTX1 belongs to the α-Ktx15 scorpion toxins family (αKTx15-4). Characterized members of this family share high sequence similarity and were found to block preferentially IA-type voltage-dependent K(+) currents in rat cerebellum granular cells in an irreversible way. In the current work, we studied the effects of native AaTX1 (nAaTX1) using whole-cell patch-clamp recordings of IA current in substantia nigra pars compacta dopaminergic neurons. At 250 nM, AaTX1 induces 90% decrease in IA current amplitude. Its activity was found to be comparable to that of rAmmTX3 (αKTx15-3), which differs by only one conserved (R/K) amino acid in the 19th position suggesting that the difference between R19 and K19 in AaTX1 and AmmTX3, respectively, may not be critical for the toxins' effects. Molecular docking of both toxins with Kv4.3 channel is in agreement with experimental data and suggests the implication of the functional dyade K27-Y36 in toxin-channel interactions. Since AaTX1 is not highly abundant in Aa venom, it was synthesized as well as AmmTX3. Synthetic peptides, native AaTX1 and rAmmTX3 peptides showed qualitatively the same pharmacological activity. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on Kv4.3 channel.

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)).

A thermoactive L-amino acid oxidase from Cerastes cerastes snake venom: purification, biochemical and molecular characterization.

A new L-amino acid oxidase (LAAO) from Cerastes cerastes snake venom, named CC-LAAO, was purified to homogeneity using a combination of size-exclusion, ion-exchange and affinity chromatography. CC-LAAO is a homodimeric glycosylated flavoprotein with a molecular mass around 58 kDa under reducing conditions and about 115 kDa in its native form when analyzed by SDS-PAGE and gel filtration chromatography, respectively. This enzyme displayed a Michaelis-Menten behavior with an optimal pH at 7.8. However, unlike known SV-LAAOs which display their maximum activity at 37 °C, CC-LAAO has an optimal temperature at 50 °C. Kinetic studies showed that the enzyme displayed high specificity towards hydrophobic L-amino acids. The best substrates were L-Phe, L-Met and L-Leu. CC-LAAO activity was inhibited by the substrate analog N-acetyl tryptophan. The N-terminal amino acid sequence of this protein was determined by automated Edman degradation. The CC-LAAO cDNA was cloned from the venom gland total RNA preparation. The cDNA sequence contained an open-reading frame (ORF) of 1551-bp, which encoded a protein of 516 amino acids comprising a signal peptide of 18 amino acids and 498-residues mature protein. CC-LAAO sequence and its tertiary model shared high similarity with other snake venom LAAOs.

Lebecin, a new C-type lectin like protein from Macrovipera lebetina venom with anti-tumor activity against the breast cancer cell line MDA-MB231.

C-type lectins like proteins display various biological activities and are known to affect especially platelet aggregation. Few of them have been reported to have anti-tumor effects. In this study, we have identified and characterized a new C-type lectin like protein, named lebecin. Lebecin is a heterodimeric protein of 30 kDa. The N-terminal amino acid sequences of both subunits were determined by Edman degradation and the entire amino acid sequences were deduced from cDNAs. The precursors of both lebecin subunits contain a 23-amino acid residue signal peptide and the mature α and ß subunits are composed of 129 and 131 amino acids, respectively. Lebecin is shown to be a potent inhibitor of MDA-MB231 human breast cancer cells proliferation. Furthermore, lebecin dose-dependently inhibited the integrin-mediated attachment of these cells to different adhesion substrata. This novel C-type lectin also completely blocked MDA-MB231 cells migration towards fibronectin and fibrinogen in haptotaxis assays.

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.

The pathological effects of Heminecrolysin, a dermonecrotic toxin from Hemiscorpius lepturus scorpion venom are mediated through its lysophospholipase D activity.

We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein responsible for the main pathological effects observed following Hemiscorpius (H.) lepturus scorpion envenomation. We aimed herein to further investigate the kinetics and molecular mechanisms triggered by Heminecrolysin to initiate hematological disorders and inflammatory reaction. We show that Heminecrolysin highly hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, with a Vmax = 1481 ± 51 µmol/min/mg and a Km = 97 ± 16.78 µM, at a much lesser extend sphingomyelin but not phosphatidylcholine substrates. Its lysophospholipase D (lysoPLD) catalytic efficiency, up to three orders of magnitude higher, comparatively to spider's SMaseDs (newly referred as phospholipases D; PLDs), could explain its strong hemolytic capacity. Chelating agents such as EDTA, EGTA, and 1, 10-phenantroline blocked Heminecrolysin-induced LPC hydrolysis at 98, 48, and 70% respectively. Hemolysis blockade occurs only when the toxin is added to erythrocytes in the presence of serum, source of LPC and complement, indicating that the production of LPA and the presence of complement are mandatory for hemolysis. Moreover, we show that Heminecrolysin efficiently binds to erythrocyte's membrane and provokes phosphatidylserine (PS) translocation without cleavage of glycophorin A, suggesting that, unlike spider's PLDs, complement was activated only via the classical pathway. Interestingly, Heminecrolysin was found to induce PS exposure on human nucleated Jurkat T cells, to stimulate secretion of the pro-inflammatory (TNF-α, IL-6), and anti-inflammatory (IL-10) cytokines by human monocytes, and to provoke a disseminated intravascular coagulation on chick embryo chorioallantoic membrane model system. Taken together, our results indicate that Heminecrolysin evokes the major characteristic clinical features of H. lepturus envenomation by using mainly its lysoPLD, rather than its SMaseD's, activity.

Antitumoral potential of Tunisian snake venoms secreted phospholipases A2.

Phospholipases type A2 (PLA2s) are the most abundant proteins found in Viperidae snake venom. They are quite fascinating from both a biological and structural point of view. Despite similarity in their structures and common catalytic properties, they exhibit a wide spectrum of pharmacological activities. Besides being hydrolases, secreted phospholipases A2 (sPLA2) are an important group of toxins, whose action at the molecular level is still a matter of debate. These proteins can display toxic effects by different mechanisms. In addition to neurotoxicity, myotoxicity, hemolytic activity, antibacterial, anticoagulant, and antiplatelet effects, some venom PLA2s show antitumor and antiangiogenic activities by mechanisms independent of their enzymatic activity. This paper aims to discuss original finding against anti-tumor and anti-angiogenic activities of sPLA2 isolated from Tunisian vipers: Cerastes cerastes and Macrovipera lebetina, representing new tools to target specific integrins, mainly, α5ß1 and αv integrins.

PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells.

A novel Kunitz-type serine proteinase inhibitor, termed PIVL, was purified to homogeneity from the venom of the Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric polypeptide chain cross-linked by three disulfide linkages with an isotope-averaged molecular mass of 7691.7 Da. The 67-residue full-length PIVL sequence was deduced from a venom gland cDNA clone. Structurally, PIVL is built by a single Kunitz/BPTI-like domain. Functionally, it is able to specifically inhibit trypsin activity. Interestingly, PIVL exhibits an anti-tumor effect and displays integrin inhibitory activity without being cytotoxic. Here we show that PIVL is able to dose-dependently inhibit the adhesion, migration and invasion of human glioblastoma U87 cells. Our results also show that PIVL impairs the function of αvß3 and to a lesser extent, the activity of αvß6, αvß5, α1ß1 and α5ß1 integrins. Interestingly, we demonstrate that the (41)RGN(43) motif of PIVL is likely responsible for its anti-cancer effect. By using time lapse videomicroscopy, we found that PIVL significantly reduced U87 cells motility and affected cell directionality persistence by 68%. These findings reveal novel pharmacological effects for a Kunitz-type serine proteinase inhibitor.

Expression of a functional recombinant C-type lectin-like protein lebecetin in the human embryonic kidney cells.

Lebecetin is an anticoagulant C-type lectin-like protein that was previously isolated from Macrovipera lebetina venom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib and to exhibit a broad spectrum of inhibitory activities on various integrin-mediated functions of tumor cells, including adhesion, proliferation, and cell migration. This study aimed to investigate the structure-function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co-localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal-chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represents a prerequisite for functioning.