Modulation of Orai1 by cationic peptides triggers their direct cytosolic uptake.

At concentrations exceeding 10 µM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake.

Correction: A FRET-based biosensor for measuring Gα13 activation in single cells.

[This corrects the article DOI: 10.1371/journal.pone.0193705.].

A FRET-based biosensor for measuring Gα13 activation in single cells.

Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gß1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.

Linear Peptides in Intracellular Applications.

To this point, efforts to develop therapeutic peptides for intracellular applications were guided by the perception that unmodified linear peptides are highly unstable and therefore structural modifications are required to reduce proteolytic breakdown. Largely, this concept is a consequence of the fact that most research on intracellular peptides hitherto has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Interestingly, inside cells, endogenous peptides lacking any chemical modifications to enhance stability escape degradation to the point that they may even modulate intracellular signaling pathways. In addition, many unmodified synthetic peptides designed to interfere with intracellular signaling, following introduction into cells, have the expected activity demonstrating that biologically relevant concentrations can be reached. This review provides an overview of results and techniques relating to the exploration and application of linear, unmodified peptides. After an introduction to intracellular peptide turnover, the review mentions examples for synthetic peptides as modulators of intracellular signaling, introduces endogenous peptides with bioactivity, techniques to measure peptide stability, and peptide delivery. Future experiments should elucidate the rules needed to predict promising peptide candidates.

Characterization of 2,3,6,7,10,11-hexahydroxytriphenylene and its effects on cell viability in human cancer cell lines.

We synthesized 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP), characterized it by electrochemistry, spectroelectrochemistry, and electron paramagnetic resonance techniques, and evaluated its cytotoxicity to human cancer cell lines. The results revealed that HHTP has accessible higher-oxidation states, especially the tris-semiquinone monoradical. This species is stable and is formed after being stored for months. HHTP exhibited cytotoxic effects on 5 human cancer cell lines, including glioma and lung cancer cells. The cytotoxic effect was evaluated based on the decrease in cell viability, increases in the percentage of cells with fragmented DNA, and elevated numbers of annexin V-PI-positive cells after HHTP treatment.

Sulfated heterorhamnans from the green seaweed Gayralia oxysperma: partial depolymerization, chemical structure and antitumor activity.

Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells.

Preparation and electrochemical characterization of a carbon ceramic electrode modified with ferrocenecarboxylic acid.

The present paper describes the characterization of a carbon ceramic electrode modified with ferrocenecarboxylic acid (designated as CCE/Fc) by electrochemical techniques and its detection ability for dopamine. From cyclic voltammetric experiments, it was observed that the CCE/Fc presented a redox pair at E(pa) = 405 mV and E(pc) = 335 mV (ΔE = 70 mV), related to the ferrocene/ferrocenium process. Studies showed a considerably increase in the redox currents at the same oxidation potential of ferrocene (E(pa) = 414 mV vs. Ag/AgCl) in the presence of dopamine (DA), differently from those observed when using only the unmodified CCE, in which the anodic peak increase was considerably lower. From SWV experiments, it was observed that the AA (ascorbic acid) oxidation at CCE/Fc occurred in a different potential than the DA oxidation (with a peak separation of approximately 200 mV). Moreover, CCE/Fc did not respond to different AA concentrations, indicating that it is possible to determine DA without the AA interference with this electrode.