Ernesto Ortiz

Scorpion venom components as potential candidates for drug development

Abstract Scorpions are well known for their dangerous stings that can result in severe consequences for human beings, including death. Neurotoxins present in their venoms are responsible for their toxicity. Due to their medical relevance, toxins have been the driving force in the scorpion natural compounds research field. On the other hand, for thousands of years, scorpions and their venoms have been applied in traditional medicine, mainly in Asia and Africa. With the remarkable growth in the number of characterized scorpion venom components, several drug candidates have been found with the potential to tackle many of the emerging global medical threats. Scorpions have become a valuable source of biologically active molecules, from novel antibiotics to potential anticancer therapeutics. Other venom components have drawn attention as useful scaffolds for the development of drugs. This review summarizes the most promising candidates for drug development that have been isolated from scorpion venoms.

Scorpion and spider venom peptides: Gene cloning and peptide expression

This communication reviews most of the important findings related to venom components isolated from scorpions and spiders, mainly by means of gene cloning and expression. Rather than revising results obtained by classical biochemical studies that report structure and function of venom components, here the emphasis is placed on cloning and identification of genes present in the venomous glands of these arachnids. Aspects related to cDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed, showing some numbers of species and components already identified, but also shortly mentioning limitations and perspectives of research for the future in this field.

Gene cloning and functional characterization of four novel antimicrobial-like peptides from scorpions of the family Vaejovidae

From the cDNA libraries made from the venom glands of two scorpions belonging to the Vaejovidae family, four different putative non disulfide-bridged antimicrobial peptides were identified: VmCT1 and VmCT2 from Vaejovis mexicanus smithi plus VsCT1 and VsCT2 from Vaejovis subcristatus. These short peptides (with only 13 amino acid residues each) share important amino acid sequence similarities among themselves and with other reported antimicrobial peptides, but their biological activities vary dramatically. This communication reports the cloning, chemical synthesis and characterization of these peptides. Two peptides, VmCT1 and VmCT2 showed broad-spectrum antibacterial activity with minimum inhibitory concentrations MICs in the range of 5-25 μM and 10-20 μM respectively, whereas their hemolytic activity at these concentrations was low. Structure-function relationships that might determine the differences in activities are discussed.

Novel α-conotoxins from Conus spurius and the α-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

α‐Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7‐type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [γ15E]SrIB) were compared to those exerted by the α4/7‐conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch‐clamp technique in mammalian cells expressing α1β1γδ, α4β2 and α3β4 nicotinic acetylcholine receptors. At high concentrations (10 µm), the peptides SrIA, SrIB and [γ15E]SrIB showed weak blocking effects only on α4β2 and α1β1γδ subtypes, but EI also strongly blocked α3β4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of α1β1γδ and α4β2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC50, 1.78 and 0.37 nm, respectively). These results suggest not only that the novel α‐conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both α1β1γδ and α4β2 nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin–receptor interaction, as well as models with which to design highly specific therapeutic drugs.

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain‐associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain‐related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the λ variable light chain (VL) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) VL protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2‐Arg25Gly), that is present in ∼25% of all amyloid‐associated λ6 light chains. Remarkably, the wild‐type 6aJL2 protein was more stable than were all known amyloidogenic κ and λ light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic λ6 protein, Jto. Conversely, the mutated 6aJL2‐R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of λ6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline‐encoded Vλ6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic. Proteins 2008.

Transcriptome Analysis of Scorpion Species Belonging to the Vaejovis Genus

Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist’s attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family.

Repeated sequences in bacterial chromosomes and plasmids: a glimpse from sequenced genomes.

To gain insight into the extent of exact DNA repeats in sequenced bacterial genomes and their plasmids, we analyzed the collection of completely sequenced bacterial genomes available at GenBank using the program Miropeats. This program draws graphical representations of exact DNA repeats in whole genomes. In this work, we present maps showing the extent and type (inverted or direct) of exact DNA repeats longer than 300 bp for the whole collection. These repeats may participate in a variety of events relevant for bacterial genome plasticity, such as amplifications, deletions, inversions, and translocations (via homologous recombination), as well as transposition. Additionally, we review recent data showing that high-frequency architectural variations in genomic structure occur at both the interspecies and interstrain levels.

Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M. tuberculosis

Five variants of human β-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. The HBDs were expressed as soluble or insoluble proteins depending on the expression system used, and the final protein yields ranged from 0.5 to 1.6 mg of peptide/g of wet weight cells, with purities higher than 90%. The recombinant HBDs demonstrated a direct correlation between antimicrobial activity and the number of basic charged residues; that is, their antimicrobial activity was as follows: HBD3-M-HBD2 > HBD3 = HBD3-M = HB2-KLK > HBD2 when assayed against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Interestingly, HBD2 had the best antimicrobial activity against the Mycobacterium tuberculosis strain H37Rv (1.5 μM) and the heterologous tandem peptide, HBD3-M-HBD2, had the best minimal inhibitory concentration (MIC) value (2.7 μM) against a multidrug resistance strain (MDR) of M. tuberculosis, demonstrating the feasibility of the use of HBDs against pathogenic M. tuberculosis reported to be resistant to commercial antibiotics.

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring λ6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda6 LCs.

Antarease-like Zn-metalloproteases are ubiquitous in the venom of different scorpion genera

BACKGROUND The venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis. METHODS The cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components. RESULTS Antarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species. CONCLUSIONS Antareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains. GENERAL SIGNIFICANCE For the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies.