Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG(neg)) and sialic acid-deficient (SA(neg)) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA(neg)>WT>GAG(neg). The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA(neg) and GAG(neg) cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG(neg) cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering

The human sperm protein PH-20 has hyaluronidase activity

AbstractThe PH-20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939-2949]. We have isolated the cDNA encoding the human PH-20 protein from a testis library. This cDNA was expressed in RK 13 cells using a vaccinia virus expression system. Cells expressing the human PH-20 protein possess hyaluronidase activity. Treatment with PI-PLC releases the hyaluronidase into the the medium with a concomitant large increase in enzymatic activity. These results demonstrate that the human PH-20 protein has hyaluronidase activity

HIV gp41 Engages gC1qR on CD4+ T Cells to Induce the Expression of an NK Ligand through the PIP3/H2O2 Pathway

CD4+ T cell loss is central to HIV pathogenesis. In the initial weeks post-infection, the great majority of dying cells are uninfected CD4+ T cells. We previously showed that the 3S motif of HIV-1 gp41 induces surface expression of NKp44L, a cellular ligand for an activating NK receptor, on uninfected bystander CD4+ T cells, rendering them susceptible to autologous NK killing. However, the mechanism of the 3S mediated NKp44L surface expression on CD4+ T cells remains unknown. Here, using immunoprecipitation, ELISA and blocking antibodies, we demonstrate that the 3S motif of HIV-1 gp41 binds to gC1qR on CD4+ T cells. We also show that the 3S peptide and two endogenous gC1qR ligands, C1q and HK, each trigger the translocation of pre-existing NKp44L molecules through a signaling cascade that involves sequential activation of PI3K, NADPH oxidase and p190 RhoGAP, and TC10 inactivation. The involvement of PI3K and NADPH oxidase derives from 2D PAGE experiments and the use of PIP3 and H2O2 as well as small molecule inhibitors to respectively induce and inhibit NKp44L surface expression. Using plasmid encoding wild type or mutated form of p190 RhoGAP, we show that 3S mediated NKp44L surface expression on CD4+ T cells is dependent on p190 RhoGAP. Finally, the role of TC10 in NKp44L surface induction was demonstrated by measuring Rho protein activity following 3S stimulation and using RNA interference. Thus, our results identify gC1qR as a new receptor of HIV-gp41 and demonstrate the signaling cascade it triggers. These findings identify potential mechanisms that new therapeutic strategies could use to prevent the CD4+ T cell depletion during HIV infection and provide further evidence of a detrimental role played by NK cells in CD4+ T cell depletion during HIV-1 infection

Effets de peptides vecteurs sur le cytosquelette d'actine après internalisation dans les cellules tumorales

Trois peptides vecteurs ont été criblés pour leur capacité à réorganiser le cytosquelette d actine dans des fibroblastes tumoraux (cellules EF). Les peptides vecteurs sont des peptides chargés positivement capables de traverser les membranes plasmiques des cellules. Dans un premier temps, une étiquette stable permettant la quantification de l internalisation et l étude du processus de dégradation de peptides vecteurs via une méthode de quantification du peptide basée sur la spectrométrie de masse MALDI-TOF a été synthétisée. Ainsi, un nouveau peptide vecteur : msr(R/W)9 est capable d entrer et peut s accumuler dans des cellules CHO. Dans une deuxième étude, nous avons testé trois peptides vecteurs sur des fibroblastes tumoraux qui n expriment pas la Zyxine, entraînant alors une désorganisation du cytosquelette d actine. Leurs effets potentiels sur la réorganisation du cytosquelette d actine et la ré-expression de la Zyxine ont été évalués à partir d études de la polymérisation d actine in vitro dans des lysats cellulaires par anisotropie de fluorescence, de la morphologie des fibroblastes par immunomarquage de l actine, de la motilité cellulaire par vidéomicroscopie et enfin de la capacité à croître en indépendance d ancrage. Les résultats obtenus montrent que ces trois peptides semblent réverser le phénotype tumoral des cellules EF alors qu ils sont inactifs sur les fibroblastes sains. Enfin, des expériences de pontage chimique entre ces trois peptides et l actine, nous ont permis de mettre en évidence une zone d interactions, identifiée et caractérisée par spectrométrie de masse, entre ces différents partenaires.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Tryptophan, an Amino-Acid Endowed with Unique Properties and Its Many Roles in Membrane Proteins

International audienceTryptophan is an aromatic amino acid with unique physico-chemical properties. It is often encountered in membrane proteins, especially at the level of the water/bilayer interface. It plays a role in membrane protein stabilization, anchoring and orientation in lipid bilayers. It has a hydrophobic character but can also engage in many types of interactions, such as π–cation or hydrogen bonds. In this review, we give an overview of the role of tryptophan in membrane proteins and a more detailed description of the underlying noncovalent interactions it can engage in with membrane partners

Tryptophan, more than just an interfacial amino acid in the membrane activity of cationic cell-penetrating and antimicrobial peptides

International audienceAbstract Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of μ- and δ-opioid receptors and an additional address subsite on the δ-receptor

International audienceDermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I

Binding and crossing: Methods for the characterization of membrane-active peptides interactions with membranes at the molecular level

International audienceAntimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling

An integrated cross-linking-MS approach to investigate cell penetrating peptides interacting partners

Cell penetrating peptides (CPPs) are attracting attention because of their ability to deliver biologically active molecules into cells. On their way they can interact with membrane and intracellular proteins. To fully understand and improve CPP efficiency as drug delivery tools, their partners need to be identified. To investigate CPP-protein complexes, chemical cross-linking coupled to mass spectrometry is a relevant method. With this aim, we developed an original approach based on two parallel strategies, an intact complex analysis and a bottom-up one, to have a global characterization of the cross-linked complexes composition as well as a detailed mapping of the interaction zones. Biological significance: The robust and efficient cross-linking-MS workflow presented here can easily be adapted to any CPP-protein interacting system and could thus contribute to a better understanding of CPPs activity as cell-specific drug delivery tools. We validated the relevancy of this cross-linking-MS approach with two biologically active CPPs, (R/W)9 and (R/W)16, and two interacting protein partners, actin and albumin, previously reported using isothermal titration calorimetry (ITC) and NMR. Cross-linking-MS results obtained on these previous studies allowed us to go further by providing a detailed mapping of the interaction zones. The identified interaction zones between actin and CPPs (R/W)9 and (R/W)16 are biologically meaningful. Two cross-linked zones [46–57] and [202–210] of actin are indeed involved in the modulation of its dynamics. Moreover, [46–57] domain has also been described as one interaction domain for thymosin β4 whose actin binding can be displaced by competition with (R/W)16 (NMR experiments)

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to δ-opioid receptor independently of receptor binding

International audienceRecent studies on the highly potent and selective δ-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the δ-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the δ-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to μ and δ-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the δ-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the δ-receptor site, nor in maintaining a δ-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionnally or structurally required to confer high δ-selectivity by preventing μ-site recognition and / or binding