T‐type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca²⁺) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G₁‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca²⁺ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.     

Asp-863 is a key residue for calcium-dependent activity of Escherichia coli RTX toxin alpha-haemolysin

alpha-Haemolysin is a protein toxin secreted by pathogenic strains of Escherichia coli and requires sub-millimolar Ca(2+) for optimum lytic activity. As a member of the so-called RTX toxin family it contains a Gly-rich, Asp-rich Ca(2+)-binding domain, consisting of a series of nonapeptides repeated in tandem. Asp-863 is located immediately after the last-but-one nonapeptide. A mutant in which Asp-863 has been substituted by Gly displays a requirement for Ca(2+) that is 100-fold higher than the wild-type. Membrane lytic activity, as well as a conformational change revealed through an increase in intrinsic fluorescence, and the appearance of Ca(2+)-bound protein monomers resolvable by fast protein liquid chromatography, are all three dependent on Ca(2+) concentrations in the 2-20 mM range. Most RTX toxins have an Asp or Glu residue located at a position homologous to Asp-863, thus the key role of this residue for Ca(2+) requirements of alpha-haemolysin may be a general feature of this family of toxins.     

Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring

The interfacial sequence DKWASLWNWFNITNWLWYIK, preceding the transmembrane anchor of gp41 glycoprotein subunit, has been shown to be essential for fusion activity and incorporation into virions. HIV(c), a peptide representing this region, formed lytic pores in liposomes composed of the main lipids occurring in the human immunodeficiency virus, type 1 (HIV-1), envelope, i.e. 1-palmitoyl-2-oleoylphosphatidylcholine (POPC):sphingomyelin (SPM):cholesterol (Chol) (1:1:1 mole ratio), at low (>1:10,000) peptide-to-lipid mole ratio, and promoted the mixing of vesicular lipids at >1:1000 peptide-to-lipid mole ratios. Inclusion of SPM or Chol in POPC membranes had different effects. Whereas SPM sustained pore formation, Chol promoted fusion activity. Even if partitioning into membranes was not affected in the absence of both SPM and Chol, HIV(c) had virtually no effect on POPC vesicles. Conditions described to disturb occurrence of lateral separation of phases in these systems reproduced the high peptide-dose requirements for leakage as found in pure POPC vesicles and inhibited fusion. Surface aggregation assays using rhodamine-labeled peptides demonstrated that SPM and Chol promoted HIV(c) self-aggregation in membranes. Employing head-group fluorescent phospholipid analogs in planar supported lipid layers, we were able to discern HIV(c) clusters associated to ordered domains. Our results support the notion that the pretransmembrane sequence may participate in the clustering of gp41 monomers within the HIV-1 envelope, and in bilayer architecture destabilization at the loci of fusion.     

His-859 is an essential residue for the activity and pH dependence of Escherichia coli RTX toxin alpha-hemolysin

Escherichia coli alpha-hemolysin (HlyA) is a toxin protein that, in common with other members of the RTX family, contains a calcium-binding domain consisting of a number of Gly- and Asp-rich nonapeptides (17 in this case) repeated in tandem. Amino acid number 6 in these nonapeptides is almost invariably Asp, and occasionally Asn, but HlyA contains a His residue (number 859 in the chain) in position 6 of the last-but-one nonapeptide. HlyA mutants have been prepared, by site-directed mutagenesis, in which His-859 has been replaced by an Asn (H859N) or by Asp (H859D). HlyA exists in aqueous media in an aggregate-monomer equilibrium, but only the monomer containing bound Ca(2+) (HlyA.Ca) appears to be competent to achieve target membrane insertion and subsequent lysis. In mutant H859N, equilibrium appears to be shifted toward the aggregate, therefore the protein does not exchange Ca(2+) with the aqueous environment, no HlyA.Ca monomers are detected, and the protein lacks any membrane lytic activity. Mutant H859D in turn is almost indistinguishable from the wild-type regarding its calcium binding and membrane lytic activity, however, it differs significantly in its pH dependence. Wild-type HlyA activity decreases sigmoidally with pH, following rather closely the protonation curve of a His residue (apparent pK(a) approximately 6.5). With mutant H859D activity decreases almost linearly with pH and to a smaller extent. It can be concluded that His-859 plays a critical role in several aspects of HlyA activity, namely self-aggregation properties, calcium binding, hemolysis, and pH dependence.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.     

Human muscle hardness assessment during incremental isometric contraction using transient elastography

The aim of this study was to investigate the relationship between biceps brachii hardness using the transient elastography technique, and its activity level by quantifying the surface electromyographic signal (sEMG). Ten healthy subjects volunteered for this protocol. To assess the maximal biceps brachii myoelectric activity (sEMG-RMSm), subjects had to achieve their maximal voluntary contraction trial during an elbow flexion effort. They were then asked to perform an isometric biceps sEMG-RMS ramp trial in elbow flexion from 0% to 50% of their sEMG-RMSm in 120 s. A low-frequency pulse was sent every 5 s during all trials by an innovative shear elasticity probe previously placed over the belly of the biceps brachii allowing the calculation of a transverse shear modulus. The main results of this study were (i) the finding of a systematic linear relationship between the biceps brachii transverse shear moduli and the corresponding sEMG-RMS values. This was not the case when plotting transverse shear modulus versus the elbow flexion torque production. Therefore, the computation of a hardness index from the slope of individual transverse shear modulus-sEMG-RMS linear relationship was enabled; (ii) It was also found that the higher is the rest shear modulus, the lower is the hardness index, indicating that the transverse shear modulus change during contraction depends on its level at rest. Therefore, this non-invasive technique could be useful in the medical field to explore deep muscles which are unreachable by classical testing methods. It could also be applied for the follow-up of neuromuscular diseases inducing stiffness changes such as in Duchenne muscular dystrophy.     

Poliovirus 2b insertion into lipid monolayers and pore formation in vesicles modulated by anionic phospholipids

Enterovirus 2B viroporin has been involved in membrane permeabilization processes occurring late during cell infection. Even though 2B lacks an obvious signal sequence for translocation, the presence of a Lys-based amphipathic domain suggests that this product bears the intrinsic capacity for partitioning into negatively charged cytofacial membrane surfaces. Pore formation by poliovirus 2B attached to a maltose-binding protein (MBP) has been indeed demonstrated in pure lipid vesicles, a fact supporting spontaneous insertion into and direct permeabilization of membranes. Here, biochemical evidence is presented indicating that both processes are modulated by phosphatidylinositol and phosphatidylserine, the main anionic phospholipids existing in membranes of target organelles. Insertion into lipid monolayers and partitioning into phospholipid bilayers were sustained by both phospholipids. However, MBP-2B inserted into phosphatidylserine bilayers did not promote membrane permeabilization and addition of this lipid inhibited the leakage observed in phosphatidylinositol vesicles. Mathematical modelling of pore formation in membranes containing increasing phosphatidylserine percentages was consistent with its inhibitory effect arising from a higher reversibility of MBP-2B surface aggregation. These results support that 2B insertion and pore-opening are mechanistically distinguishable events modulated by the target membrane anionic phospholipids.     

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.