Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Beta-KTx14.3, a scorpion toxin, blocks the human potassium channel KCNQ1.

Potassium channels play a key role in regulating many physiological processes, thus, alterations in their proper functioning can lead to the development of several diseases. Hence, the search for compounds capable of regulating the activity of these channels constitutes an intense field of investigation. Potassium scorpion toxins are grouped into six subfamilies (α, ß, γ, κ, δ, and λ). However, experimental structures and functional analyses of the long chain ß-KTx subfamily are lacking. In this study, we recombinantly produced the toxins TcoKIK and beta-KTx14.3 present in the venom of Tityus costatus and Lychas mucronatus scorpions, respectively. The 3D structures of these ß-KTx toxins were determined by nuclear magnetic resonance. In both toxins, the N-terminal region is unstructured, while the C-terminal possesses the classic CSα/ß motif. TcoKIK did not show any clear activity against frog Shaker and human KCNQ1 potassium channels; however, beta-KTx14.3 was able to block the KCNQ1 channel. The toxin-channel interaction mode was investigated using molecular dynamics simulations. The results showed that this toxin could form a stable network of polar-to-polar and hydrophobic interactions with KCNQ1, involving key conserved residues in both molecular partners. The discovery and characterization of a toxin capable of inhibiting KCNQ1 pave the way for the future development of novel drugs for the treatment of human diseases caused by the malfunction of this potassium channel. STATEMENT OF SIGNIFICANCE: Scorpion toxins have been shown to rarely block human KCNQ1 channels, which participate in the regulation of cardiac processes. In this study, we obtained recombinant beta-KTx14.3 and TcoKIK toxins and determined their 3D structures by nuclear magnetic resonance. Electrophysiological studies and molecular dynamics models were employed to examine the interactions between these two toxins and the human KCNQ1, which is the major driver channel of cardiac repolarization; beta-KTx14.3 was found to block effectively this channel. Our findings provide insights for the development of novel toxin-based drugs for the treatment of cardiac channelopathies involving KCNQ1-like channels.

Novel Blocker of Onco SK3 Channels Derived from Scorpion Toxin Tamapin and Active against Migration of Cancer Cells.

Peptide-based therapy against cancer is a field of great interest for biomedical developments. Since it was shown that SK3 channels promote cancer cell migration and metastatic development, we started using these channels as targets for the development of antimetastatic drugs. Particularly, tamapin (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known. Considering this fact, we designed diverse tamapin mutants based on three different hypotheses to discover a new potent molecule to block SK3 channels. We performed in vitro studies to evaluate this new toxin derivative inhibitor of cancer cell migration. Our results can be used to generate a new tamapin-based therapy against cancer cells that express SK3 channels.

Solution structure of the inhibitor of cysteine proteases 1 from Entamoeba histolytica reveals a possible auto regulatory mechanism.

The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven ß-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.

The three-dimensional structure of the toxic peptide Cl13 from the scorpion Centruroides limpidus.

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known ß-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/ß (CSα/ß) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel ß-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.

Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress.

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

Hepta-, hexa-, penta-, tetra-, and trisaccharide resin glycosides from three species of Ipomoea and their antiproliferative activity on two glioma cell lines.

Six new partially acylated resin glycosides were isolated from convolvulin of Ipomoea purga, Ipomoea stans, and Ipomoea murucoides (Convolvulaceae). The structures of compounds 1-6 were elucidated by a combination of NMR spectroscopy and mass spectrometry. The structure of jalapinoside B (1) consists of a hexasaccharide core bonded to an 11-hydroxytetradecanoic (convolvulinic) acid forming a macrolactone acylated by a 2-methylbutanoyl, a 3-hydroxy-2-methylbutanoyl, and a quamoclinic acid B units. Purginoic acid A (2) contains a hexasaccharide core bonded to a convolvulinic acid acylated by a 3-hydroxy-2-methylbutanoyl unit. Stansin A (4) is an ester-type heterodimer, and consists of two stansoic acid A (3) units, acylated by 2-methylbutanoic and 3-hydroxy-2-methylbutanoic acids. The site of lactonization was located at C-3 of Rhamnose, and the position for the ester linkage of the monomeric unit B on the macrolactone unit A was established as C-4 of the terminal rhamnose. Compounds 5 and 6 are glycosidic acids. Murucinic acid II (5) is composed of a pentasaccharide core bonded to an 11-hydroxyhexadecanoic (jalapinolic) acid, acylated by an acetyl unit. Stansinic acid I (6) is a tetrasaccharide core bonded to a jalapinolic acid, acylated by 2-methylbutanoyl and 3-hydroxy-2-methylbutanoyl units. Preliminary testing showed the cytotoxicity of compounds 1-6 toward OVCAR and UISO-SQC-1 cancer cell lines. In addition, compound 1 showed an antiproliferative activity on glioma C6 and RG2 tumor cell lines. Copyright © 2016 John Wiley & Sons, Ltd.

Solution structure and antiparasitic activity of scorpine-like peptides from Hoffmannihadrurus gertschi.

Scorpine-like peptides are two domain peptides found in different scorpion venoms displaying various antimicrobial, cytolytic, and potassium channel-blocking activities. The relative contribution of each domain to their different activities remains to be elucidated. Here, we report the recombinant production, solution structure, and antiparasitic activity of Hge36, first identified as a naturally occurring truncated form of a Scorpine-like peptide from the venom of Hoffmannihadrurus gertschi. We also show that removing the first four residues from Hge36 renders a molecule with enhanced potassium channel-blocking and antiparasitic activities. Our results are important to rationalize the structure-function relationships of a pharmacologically versatile molecular scaffold.

Cytotoxicity of recombinant tamapin and related toxin-like peptides on model cell lines.

The scorpion toxin tamapin displays the most potent and selective blockage against KCa2.2 channels known to date. In this work, we report the biosynthesis, three-dimensional structure, and cytotoxicity on cancer cell lines (Jurkat E6-1 and human mammary breast cancer MDA-MB-231) of recombinant tamapin and five related peptides bearing mutations on residues (R6A,R7A, R13A, R6A-R7A, and GS-tamapin) that were previously suggested to be important for tamapin's activity. The indicated cell lines were used as they constitutively express KCa2.2 channels. The studied toxin-like peptides displayed lethal responses on Jurkat T cells and breast cancer cells; their effect is dose- and time-dependent with IC50 values in the nanomolar range. The order of potency is r-tamapin>GS-tamapin>R6A>R13A>R6A-R7A>R7A for Jurkat T cells and r-tamapin>R7A for MDA-MB-231 breast cancer cells. Our structural determination by NMR demonstrated that r-tamapin preserves the folding of the αKTx5 subfamily and that neither single nor double alanine mutations affect the three-dimensional structure of the wild-type peptide. In contrast, our activity assays show that changes in cytotoxicity are related to the chemical nature of certain residues. Our results suggest that the toxic activity of r-tamapin on Jurkat and breast cancer cells could be mediated by the interaction of charged residues in tamapin with KCa2.2 channels via the apoptotic cell death pathway.

Anticonvulsant and neuroprotective effects of oligosaccharides from Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (Higher Basidiomycetes).

An oligosaccharide fraction isolated from the mycelium of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (GLOS) was separated by size-exclusion chromatography. The chemical structure of GLOS consists of a disaccharide repeating unit [-4-ß-1-Galf(1-6)-O-(ß-Glcp)-1-]n (n=3,4). In addition, this study was undertaken to determine the possible anticonvulsant and neuroprotective effects of GLOS (10-80 mg/kg) on kainic acid (KA)-induced seizures. The behavioral alterations and histopathology of hippocampal neurons were studied. Our results show that GLOS inhibited convulsions in rats from KA-induced seizures, reduced the degeneration pattern in the CA3 region of rats, decreased astrocytic reactivity, and reduced the expression of IL-1ß and TNF-α induced by KA. These results indicate a potential anticonvulsant and neuroprotective effects of GLOS.

Structure, function, and chemical synthesis of Vaejovis mexicanus peptide 24: a novel potent blocker of Kv1.3 potassium channels of human T lymphocytes.

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/ß motif consisting of a single-turn α-helix and a three-stranded antiparallel ß-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 sodium channels.

The three-dimensional structures of the long-chain mammalian scorpion ß-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/ß fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.

New tricks of an old pattern: structural versatility of scorpion toxins with common cysteine spacing.

Scorpion venoms are a rich source of K(+) channel-blocking peptides. For the most part, they are structurally related small disulfide-rich proteins containing a conserved pattern of six cysteines that is assumed to dictate their common three-dimensional folding. In the conventional pattern, two disulfide bridges connect an α-helical segment to the C-terminal strand of a double- or triple-stranded ß-sheet, conforming a cystine-stabilized α/ß scaffold (CSα/ß). Here we show that two K(+) channel-blocking peptides from Tityus scorpions conserve the cysteine spacing of common scorpion venom peptides but display an unconventional disulfide pattern, accompanied by a complete rearrangement of the secondary structure topology into a CS helix-loop-helix fold. Sequence and structural comparisons of the peptides adopting this novel fold suggest that it would be a new elaboration of the widespread CSα/ß scaffold, thus revealing an unexpected structural versatility of these small disulfide-rich proteins. Acknowledgment of such versatility is important to understand how venom structural complexity emerged on a limited number of molecular scaffolds.

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.