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.

Dynamics underlie the drug recognition mechanism by the efflux transporter EmrE.

The multidrug efflux transporter EmrE from Escherichia coli requires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket.

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.

Sensory and memory processing in old female and male Wistar rat brain, and its relationship with the cortical and hippocampal redox state.

The brain is one of the most sensitive organs damaged during aging due to its susceptibility to the aging-related oxidative stress. Hence, in this study, the sensory nerve pathway integrity and the memory were evaluated and related to the redox state, the antioxidant enzymes function, and the protein oxidative damage in the brain cortex (Cx) and the hippocampus (Hc) of young (4-month-old) and old (24-month-old) male and female Wistar rats. Evoked potentials (EP) were performed for the auditory, visual, and somatosensory pathways. In both males and females, the old rat groups' latencies were larger in almost all waves when compared to the young same-sex animals. The novel object test was performed to evaluate memory. The superoxide dismutase and catalase antioxidant activity, as well as the protein oxidative damage, and the redox state were evaluated. Magnetic resonance (MR) imaging was used to obtain the diffusion tensor imaging, and the brain volume, while MR spectroscopy was used to obtain the brain metabolite concentrations (glutamine, glutamate, Myo-inositol, N-acetyl-aspartate, creatine) in the Cx and the Hc of young and old females. Our data suggest that, although there are limited variations regarding memory and nerve conduction velocity by sex, the differences concerning the redox status might be important to explain the dissimilar reactions during brain aging between males and females. Moreover, the increment in Myo-inositol levels in the Hc of old rats and the brain volume decrease suggest that redox state alterations might be correlated to neuroinflammation during brain aging.

Increase of CaV3 channel activity induced by HVA ß1b-subunit is not mediated by a physical interaction.

OBJECTIVE: Low voltage-activated (LVA) calcium channels are crucial for regulating oscillatory behavior in several types of neurons and other excitable cells. LVA channels dysfunction has been implicated in epilepsy, neuropathic pain, cancer, among other diseases. Unlike for High Voltage-Activated (HVA) channels, voltage-dependence and kinetics of currents carried by recombinant LVA, i.e., CaV3 channels, are quite similar to those observed in native currents. Therefore, whether these channels are regulated by HVA auxiliary subunits, remain controversial. Here, we used the α1-subunits of CaV3.1, CaV3.2, and CaV3.3 channels, together with HVA auxiliary ß-subunits to perform electrophysiological, confocal microscopy and immunoprecipitation experiments, in order to further explore this possibility. RESULTS: Functional expression of CaV3 channels is up-regulated by all four ß-subunits, although most consistent effects were observed with the ß1b-subunit. The biophysical properties of CaV3 channels were not modified by any ß-subunit. Furthermore, although ß1b-subunits increased colocalization of GFP-tagged CaV3 channels and the plasma membrane of HEK-293 cells, western blots analysis revealed the absence of physical interaction between CaV3.3 and ß1b-subunits as no co-immunoprecipitation was observed. These results provide solid evidence that the up-regulation of LVA channels in the presence of HVA-ß1b subunit is not mediated by a high affinity interaction between both proteins.

The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity.

Voltage-gated sodium (NaV) channels have been related with cell migration and invasiveness in human cancers. We previously reported the contribution of NaV1.6 channels activity with the invasion capacity of cervical cancer (CeCa) positive to Human Papilloma Virus type 16 (HPV16), which accounts for 50% of all CeCa cases. Here, we show that NaV1.6 gene (SCN8A) overexpression is a general characteristic of CeCa, regardless of the HPV type. In contrast, no differences were observed in NaV1.6 channel expression between samples of non-cancerous and cervical intraepithelial neoplasia. Additionally, we found that CeCa cell lines, C33A, SiHa, CaSki and HeLa, express mainly the splice variant of SCN8A that lacks exon 18, shown to encode for an intracellularly localized NaV1.6 channel, whereas the full-length adult form was present in CeCa biopsies. Correlatively, patch-clamp experiments showed no evidence of whole-cell sodium currents (INa) in CeCa cell lines. Heterologous expression of full-length NaV1.6 isoform in C33A cells produced INa, which were sufficient to significantly increase invasion capacity and matrix metalloproteinase type 2 (MMP-2) activity. These data suggest that upregulation of NaV1.6 channel expression occurs when cervical epithelium have been transformed into cancer cells, and that NaV1.6-mediated invasiveness of CeCa cells involves MMP-2 activity. Thus, our findings support the notion about using NaV channels as therapeutic targets against cancer metastasis.

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.