Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization

Des protéines de synthèse taillées sur mesure pour investiguer le vivant

International audienceDepuis la synthèse des premiers dipeptides par Curtius 1 et Fisher 2 , les protéines ont constitué un objectif fascinant pour des générations de chimistes. Cinquante ans de développements méthodologiques ont été nécessaires pour aboutir en 1954 à la synthèse d'une hormone bioactive de 9 acides aminés (aa), l'ocytocine (Fig. 1). 3 En 1971, la synthèse d'une enzyme, la ribonucléase A (123 aa), a été réalisée par Merrifield 4 en enchaînant 246 réactions sur un support insoluble. 5 Ce tour de force a démontré la grande efficacité de la synthèse de peptides en phase solide (SPPS), qui est aujourd'hui un outil classique pour la synthèse automatisée de peptides composés de quelques dizaines d'acides aminés. Le dernier jalon marquant de cette épopée scientifique est l'invention du concept de « ligation chimique », ou des réactions chimiques sélectives sont mises en oeuvre en milieu aqueux pour lier des segments non-protégés obtenus par SPPS ou biotechnologie. Introduit en 1992 pour la synthèse d'une protéine dont le squelette est modifié, 6 il est adapté deux ans plus tard à une version « native » où une liaison peptidique est formée au site de jonction (Native Chemical Ligation, NCL, 7,8 Fig. 2A). 25 ans après sa découverte, cette réaction reste la plus utilisée pour la synthèse de protéines. 9 Elle a permis l'accès à des cibles de plus de 300 acides aminés 10,11 et a ouvert une voie inédite pour la synthèse parfaitement contrôlée de protéines natives ou modifiées spécifiquement au niveau de leur squelette ou de certaines chaînes latérales. En ce sens, l'approche « chimique » de la synthèse de protéines vient compléter les méthodes biotechnologiques exploitant des systèmes vivants, avec l'avantage de pouvoir créer des outils sur-mesure pour décrypter le vivant à une résolution atomique

A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics

Abstract Most eukaryotic RNA regulators recognise their RNA and protein partners by the combinatorial use of several RNA binding domains. Inter-domain dynamics and interactions play a key role in recognition and can be analysed by techniques such as NMR or FRET, provided that the information relative to the individual interactions can be de-convoluted. Segmentally labelling the proteins by ligating labelled and unlabelled peptide chains allows one to filter out unwanted information and observe the labelled moieties only. Several strategies have been implemented to ligate two protein fragments, but multiple ligations, which are necessary to segmentally label proteins of more than two domains, are more challenging and often dependent on the structure and solubility of the domains. Here we report a method to ligate multiple protein segments that allows the fast, high yield labelling of both internal and end domains, depending on the requirements. We use TCEP and mercaptophenylacetic acid (MPAA) in an optimised reaction environment to achieve an efficient ligation of protein domains independently from their structure or solubility. We expect the method will provide a useful tool for the molecular study of combinatorial protein–RNA recognition in RNA regulation

In situ thioester formation for protein ligation using a-methylcysteine

International audienceThe progress of total chemical protein synthesis has been hampered by difficulties in preparing peptide thioesters by standard Fmoc peptide synthesis. The amino acid, a-methylcysteine, sited at the C-terminus of a peptide can substitute for a thioester in peptide ligation reactions. C-terminal a-methylcysteine is fully compatible with Fmoc peptide synthesis and its use in ligation is very simple and robust. Its potential is demonstrated with the synthesis of model proteins

Head to tail cyclisation of cell-penetrating peptides: impact on GAG-dependent internalisation and direct translocation

International audienceA series of cyclic lipidated oligo-Arg cell penetrating peptides were synthesised with varied macrocycle size and lipid chain anchoring site. The study of their cellular uptake revealed different structural requirements to promote efficient glycosaminoglycan-dependent endocytosis and direct translocation

Dosage et pistage de peptides Troyens dans les cellules

Les peptides Troyens ou vecteurs sont capables de passer les membranes biologiques et de véhiculer des principes actifs dans le cytoplasme ou le noyau des cellules dans lesquelles ils sont entrés. Les méthodes indirectes utilisées jusqu'à présent pour détecter ces peptides dans les cellules n'ont pas permis d'établir de manière univoque le(s) mécanisme(s) de leur internalisation. La méthode de quantification, basée sur la spectrométrie de masse MALDI-TOF, que nous avons mise au point pour quantifier ces peptides Troyens dans les cellules est développée dans cette revue

Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles - A straightforward Screening Assay for Investigating the Internalization Mechanism.

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The sequence Pro(295)-Thr(311) of the hinge region of oestrogen receptor alpha is involved in ERK1/2 activation via GPR30 in leiomyoma cells

International audienceThe ER alpha (oestrogen receptor alpha)-derived peptide ER alpha 17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the G alpha(i) protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of beta-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ER alpha is down-regulated by prolonged treatment with E-2 (oestradiol) or specific ER alpha siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ER alpha is required for the action of ER alpha 17p. In addition, its PLM sequence, which interferes with the formation of the ER alpha-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ER alpha 17p is, to our knowledge, the first known peptide targeting ER alpha-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects

Label-free measurement of histone lysine methyltransferases activity by MALDI-TOF mass spectrometry.

International audience: Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive-labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS based assay for detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors

ERα17p, a peptide reproducing the hinge region of the estrogen receptor α associates to biological membranes: A biophysical approach

Recently, we identified a peptide (ERα17p, P 295LMIKRSKKNSLALSLT311) that corresponds to the 295-311 sequence of the estrogen receptor α (ERα, hinge region) and which exerts a panel of pharmacological effects in breast cancer cells. Remarkably, these effects can result from the interaction of ERα17p with the plasma membrane. Herein, we show that ERα17p adopts a β-sheet secondary structure when in contact with anionic phospholipids and that it is engulfed within the lipid bilayer. While ERα17p increases the fluidity of membrane mimics, it weakly internalizes in living cells. In light of the above, one may evoke one important role of the 295-311 region of the ERα: the corresponding peptide could be secreted/delivered to the extracellular medium to interact with neighboring cells, both intracellularly and at the membrane level. Finally, the 295-311 region of ERα being in proximity to the cystein-447, the palmitoylation site of the ERα raises the question of its involvement in the interaction/stabilization of the protein with the membrane. © 2011 Elsevier Inc. All rights reserved.SCOPUS: cp.jinfo:eu-repo/semantics/publishe