Laurent Chaloin

Translocating peptides and proteins and their use for gene delivery

A dramatic surge in the development of peptides for gene delivery in vitro and in vivo has been witnessed in the past decade. A better understanding of the structural and mechanistic properties of peptides has been an important step for the rational design of optimal peptide-based gene delivery systems. Research has focused on the design of short synthetic peptides that overcome both extracellular and intracellular limitations of other gene delivery systems by binding reversibly and condensing DNA, specifically targeting cells and/or tissues, rapidly releasing plasmids into the cytoplasm and mediating efficient nuclear translocation.

A New Potent HIV-1 Reverse Transcriptase Inhibitor A SYNTHETIC PEPTIDE DERIVED FROM THE INTERFACE SUBUNIT DOMAINS

The biologically relevant and active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase found in infectious virions are heterodimers produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. We have shown that the dimeric nature of reverse transcriptase represents a important target for the design of a new class of antiviral agents. In this work, we propose a new strategy for its inhibition by targeting protein/protein interactions during viral formation in infected cells. From the screening of peptides derived from the tryptophan cluster at the interface of the connection subdomain, we have designed a short peptide (10 residues) corresponding to residues 395–404, which can block dimerization of reverse transcriptase in vitro and in infected cells. This peptide is highly efficient in abolishing the production of viral particles, without any adverse toxic side effects, when transduced into human immunodeficiency virus type 1-infected cells together with a new peptide carrier.

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression.

The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNA-pPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNA-pPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipid-based methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.

Detection of Peptide-Lipid Interactions in Mixed Monolayers, Using Isotherms, Atomic Force Microscopy, and Fourier Transform Infrared Analyses

To improve the understanding of the membrane uptake of an amphipathic and positively charged vector peptide, we studied the interactions of this peptide with different phospholipids, the nature of whose polar headgroups and physical states were varied. Three lipids were considered: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and dioleoylphosphatidylglycerol (DOPG). The approach was carried out by three complementary methods: compression isotherms of monolayers and atomic force microscopy observations associated with Fourier transform infrared investigations. From analysis of the compression isotherms, it was concluded that the peptide interacts with all lipids and with an expansion of the mean molecular area, implying that both components form nonideal mixtures. The expansion was larger in the case of DOPG than for DPPC and DPPG because of an alpha to beta conformational transition with an increase in the peptide molar fraction. Atomic force microscopy observations showed that the presence of small amounts of peptide led to the appearance of bowl-like particles and that an increase in the peptide amounts generated the formation of filaments. In the case of DOPG, filaments were found at higher peptide molar fractions than already observed for DOPC because of the presence of negatively charged lipid headgroups.

VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability

BackgroundThe machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined.ResultsHere, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity.ConclusionDefects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.

Virus assembly and plasma membrane domains: which came first?

Viral assembly is a key step in the virus life cycle. In this review, we focus mainly on the ability of retroviruses, especially HIV-1, to assemble at the plasma membrane of their host cells. The assembly process of RNA enveloped viruses necessitates a fine orchestration between the different viral components and specific interactions between viral proteins and lipids of the host cell membrane. Searching for a comparison with another RNA enveloped virus, we refer to influenza virus to show how it could share (or not) some common features with HIV-1 assembly since both viruses are believed to assemble mainly in raft microdomains. We also discuss the role of RNA and the cellular actin cytoskeleton in enhancing these viral assembly processes. Finally, based on the literature and on new results we have obtained by molecular docking, we propose another mechanism for HIV-1 assembly in membrane domains. This mechanism involves the trapping of acidic lipids by the viral Gag protein by means of ionic protein-lipid interactions, inducing thereby formation of acidic lipid-enriched microdomains (ALEM).

Interactions of Primary Amphipathic Vector Peptides with Membranes. Conformational Consequences and Influence on Cellular Localization

Abstract. The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, α-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of β-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.

Lipid-Induced Organization of a Primary Amphipathic Peptide: A Coupled AFM-Monolayer Study

Abstract. To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a β-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate.

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing.

Ionic channels formed by a primary amphipathic peptide containing a signal peptide and a nuclear localization sequence

The peptide SP-NLS (Ac-Met-Gly-Leu-Gly-Leu-His-Leu-Leu-Leu-Ala10-Ala-Ala-Leu-Gln-Gly- Ala -Lys-Lys-Lys-Arg20-Lys-Val-NH-CH2-CH2-SH) is composed of a hydrophobic signal sequence (SP, Met-1 to Ala-16) followed by a polycationic nuclear localization sequence (NLS, Lys-17 to Val-22) terminated by a cysteamide group. Designed to act as drug carrier this primary amphipathic peptide proved cytotoxic and bactericidal when used at high concentrations, probably by inducing the formation of ion channels. In this work, we show that indeed SP-NLS exhibits a pore-forming activity when incorporated into planar lipid bilayers and Xenopus laevis oocyte plasma membranes, with conductance values of 25 pS in 0.1 M NaCl. In both membranes, the insertion of the peptide was voltage-triggered whereas the induced conductances proved almost voltage-independent. Moreover, SP-NLS ion channels were selective for monovalent cations (K+>Na+>Li+>tetraethylammonium+>choline+). The ion channel activity of this type of peptides thus provides some insight on their toxicity but also on the mechanism involved for their membrane crossing process.