Tamás Letoha

In Vitro and in Vivo Nuclear Factor-κB Inhibitory Effects of the Cell-Penetrating Penetratin Peptide

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-κB (NF-κB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-κB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-κB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IκB degradation and subsequent nuclear import of NF-κB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons.

Human scotopic spatiotemporal sensitivity: A comparison of psychophysical and electrophysiological data

The aim of the study was to investigate spatiotemporal visual functions under scotopic and photopic conditions in order to acquire human psychophysical and electrophysiological data that are comparable with contrast sensitivities based on single-unit recordings in animal experiments. Static and dynamic contrast sensitivities (CSs) and steady-state visual evoked potentials (VEPs) were measured under photopic and scotopic conditions in healthy volunteers. The results from the CS experiment indicated that the inclusion of temporal modulation and the application of scotopic luminance levels uniformly resulted in a relatively increased sensitivity for low spatial frequencies. Similarly, analysis of the second harmonic component of the VEPs demonstrated a shift from band-pass to low-pass functions. These results suggest that, under scotopic conditions, human visuospatial processing is characteristically predominated by the functional activity of the magnocellular pathways.

IN VITRO AND IN VIVO NF-κB INHIBITORY EFFECTS OF THE CELL-PENETRATING PENETRATIN PEPTIDE

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-κB (NF-κB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-κB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-κB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IκB degradation and subsequent nuclear import of NF-κB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

Contribution of syndecans to lipoplex-mediated gene delivery

The long awaited breakthrough of gene therapy significantly depends on the in vivo efficiency of targeted intracellular delivery. Hidden details of cellular uptake present a great hurdle for non-viral gene delivery with liposomes. Growing scientific evidence supports the involvement of polyanionic cell surface carbohydrates in cellular internalization of cationic liposomes. Syndecans, a highly conserved family of transmembrane heparan sulfate proteoglycans serve attachment sites for great variety of cationic ligands including growth factors, cytokines and even parasites. In the present study we quantitatively measured the contribution of various syndecan isoforms to liposome-mediated gene transfer. The obtained data show the superiority of syndecan-4, the ubiquitously expressed isoform of the syndecan family, in cellular uptake of liposomes. Applied mutational analysis demonstrated that gene delivery could be abolished by mutating the glycosaminoglycan attachment site of syndecans, highlighting the importance of polyanionic heparan sulfate side chains in the attachment of cationic liposomes. Blocking sulfation of syndecans also diminished gene delivery, a finding that confirms the essential role of polyanionic charges in binding cationic liposomes. Mutating other parts of the syndecan extracellular domain, including the cell-binding domain, had clearly smaller effect on liposome internalization. Mutational analyses also revealed that superiority of syndecan-4 in liposome-mediated gene delivery is significantly influenced by its cytoplasmic domain that orchestrates signaling pathways leading to macropinocytosis. In summary our study present a mechanistic insight into syndecan-mediated macropinocytic uptake of lipoplexes and highlights syndecan-4 as a superior target for cationic liposomes.

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction–dependent manner

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.