A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8-10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.     

HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Delta disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to <10% of its normal cellular concentration. Other than Acc1p, the N-terminal sequence of Hfa1p comprises a canonical mitochondrial targeting signal together with a matrix protease cleavage site. Accordingly, the HFA1-encoded protein was specifically assigned by Western blotting of appropriate cell fractions to the mitochondrial compartment. Removal of the mitochondrial targeting sequence abolished the competence of HFA1 DNA to complement hfal null mutants. Conversely and in contrast to the intact HFA1 sequence, the signal sequence-free HFA1 gene complemented the mutational loss of cytoplasmic acetyl-CoA carboxylase. Expression of HFA1 under the control of the ACC1 promoter restored cellular ACC activity in ACC1-defective yeast mutants to wild type levels. From this finding, it is concluded that HFA1 encodes a specific mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for intraorganellar fatty acid and, in particular, lipoic acid synthesis.     

Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.     

Analysis of the type IV secretion system-dependent cell motility of Helicobacter pylori-infected epithelial cells

The pathogenesis of Helicobacter pylori-associated disorders is strongly dependent on a specialized type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). Cytotoxin-associated gene A (CagA) is the only known H. pylori protein translocated into the host cell followed by tyrosine phosphorylation through host protein kinases. H. pylori induces cellular processes which are either PAI- or CagA-dependent (e.g., cell motility), PAI-dependent, but CagA-independent (e.g., interleukin-8 release), or PAI- and CagA-independent (e.g., cyclooxygenase-2 release). Here, we investigated H. pylori strains mutated in single PAI genes of the wild type strain Hp26695 and their effects on cell motility. We found 17 gene products out of 27 PAI genes playing a superordinated role and five PAI-encoded proteins exhibiting a clearly critical role in motogenic host cell responses, whereas the remaining five PAI gene products had no significant influence on the motogenic response in reaction to H. pylori infection. This study clearly demonstrated that H. pylori-induced cell motility and invasive growth involve type IV secretion system-dependent signalling as well as translocated and phosphorylated CagA. These findings reveal a deeper insight in to the meaning of the T4SS of H. pylori for host cell motility.     

Screening of a large panel of gastrointestinal peptide plasma levels is not adapted for the evaluation of digestive damage following irradiation

The aim of this study was to assess the potential of gastrointestinal peptide plasma levels as biomarkers of radiation-induced digestive tract damage. To this end, plasma levels of substance P, GRP, motilin, PYY, somatostatin-28, gastrin, and neurotensin were followed for up to 5 days in pigs after a 16-Gy whole-body X-irradiation, completed by a histopathological study performed at 5 days. Each peptide gave a specific response to irradiation. The plasma levels of GRP and substance P were not modified by irradiation exposure; neither were those of motilin and PYY. Concerning gastrin, a 2-3-fold increase of plasma concentration was observed in pig, which presented the most important histological alterations of the stomach. The plasma levels of somatostatin, unchanged from 1 to 4 days after irradiation, was also increased by 130% at 5 days. In contrast, a diminution of neurotensin plasma levels was noted, firstly at 1 day (-88%), and from 3 days after exposure (-50%). The present study suggested that changes in gastrin and neurotensin plasma levels were associated with structural alterations of the stomach and ileum, respectively, indicating that they may be relevant biological indicators of radiation-induced digestive damage to these segments.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La(3+) or Gd(3+), whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La(3+) or Gd(3+) at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La(3+), but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu(543) and Glu(595)/Glu(598), situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu(595)/Glu(598) in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La(3+) and Gd(3+) have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La(3+) at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.     

Parathyroid hormone-related peptide improves contractile function of stunned myocardium in rats and pigs

The effect of synthetic parathyroid hormone (PTH)-related peptide [PTHrP(1-34)] on regional myocardial function was studied in 11 anesthetized pigs. Intracoronary infusion of PTHrP (cumulative dose: 14 +/- 1 microg) decreased coronary resistance to 33 +/- 2% of baseline (P < 0.05) and regional myocardial function to 90 +/- 3% of baseline (not significant). Ischemia-reperfusion alters the activity of several kinases and therefore possibly the myocardial effects of PTHrP. In stunned myocardium, induced by 20-min ischemia and 30-min reperfusion, the dose of PTHrP reducing coronary resistance to a minimum of 29 +/- 2% was decreased to 8 +/- 2 microg (P < 0.05). Regional myocardial function was no longer decreased but increased to 132 +/- 9% (P < 0.05). The increase in regional myocardial function during PTHrP was inversely related to baseline function at 30-min reperfusion in vivo (r = 0.9) as well as in myocytes isolated from stunned pig hearts (r = 0.7). In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, blockade of endogenous PTHrP by d-Trp(12)-Tyr(34)-PTH(7-34) attenuated the recovery of left ventricular developed pressure by 30 +/- 14% (P < 0.05). Thus endogenous and exogenous PTHrP impact on the function of stunned myocardium.