Eric Vives

A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an α-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the α-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nm. In contrast, a peptide with a full α-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 °C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.

Selective coupling of a highly basic peptide to an oligonucleotide

Abstract A highly basic peptide (net charge +8) derived from the HIV-1 Tat protein is conjugated with quantitative yield within minutes to a 19 mer rhodamine-labelled phosphodiester oligonucleotide activated by the pyridine sulfenyl group. To avoid precipitation due to antagonist charges of the oligonucleotide and the peptide the conjugation was performed with high salt concentration (400mM) and acetonitrile (40%).

Lethal neurotoxicity in mice of the basic domains of HIV and SIV Rev proteins Study of these regions by circular dichroism

We have recently reported a basic domain‐mediated neurotoxic activity of HIV‐1 Tat [1991, J. Virol. 65, 961–965]. Here we have tested the neurotoxicity in vivo of several Rev‐related synthetic peptides and found that only those mimicking the basic regions of Rev from HIV‐1, HIV‐2 and SIV were lethal to mice. In contrast, the homologous domain of HTLV‐1 Rex was found to be inactive for lethal activity. Analysis of the tropism of these peptides for phospholipids has demonstrated a direct interaction of the basic domain‐containing peptides, except Rex, with acidic — but not neutral — phospholipids. As determined by circular dichroism, a possible correlation between the conformation of the basic regions and the toxicity is discussed.

One-pot labeling and purification of peptides and proteins with fluorescein maleimide

Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo

Abstract Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro‐inverso amphipathic RICK peptide as novel non‐covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self‐assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤ 20%) of the NPs did not influence their cellular internalization capacity as well as their knock‐down specificity (over‐expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one‐cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra‐cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG‐RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy‐to‐handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi‐based cancer therapeutics. Graphical abstract Figure. No Caption available.

Use of the Salmonella MgtR peptide as an antagonist of the Mycobacterium MgtC virulence factor

BACKGROUND The MgtC virulence factor has been proposed as an attractive target for antivirulence strategies because it is shared by several important bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis (Mtb). AIM A natural antagonistic peptide, MgtR, which interacts with MgtC and modulates its stability, has been identified in Salmonella, and we investigated its efficiency to target MgtC in another pathogen. MATERIALS & METHODS We evaluated the interaction between Salmonella MgtR peptide and the Mtb MgtC protein using an in vivo bacterial two-hybrid system and we addressed the effect of exogenously added synthetic MgtR and endogenously expressed peptide. RESULTS MgtR peptide strongly interacted with Mtb MgtC protein and exogenously added synthetic MgtR peptide-reduced Mtb MgtC level and interfered with the dimerization of Mtb MgtC. Importantly, heterologous expression of MgtR in Mycobacterium bovis BCG resulted in increased phagocytosis and reduced intramacrophage survival. CONCLUSION MgtR peptide can target Mtb MgtC protein and reduce mycobacterial macrophage resistance, thus providing a promising new scaffold for the development of antivirulence compounds.

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication

Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties.

Peptide-Mediated Delivery of Antisense Oligonucleotides and Related Material

Despite their simple conceptual basis, antisense strategies turned out more difficult to implement than initially anticipated. Overwhelming enthusiasm in academic laboratories and in the biotechnology industry in the early 1990s has been followed by a wave of skepticism about the real potential of nucleic acidsbased drugs. An antisense oligonucleotide (ON)-based drug has been approved for the treatment of ocular cytomegalovirus infection, and several clinical trials are now well advanced for the treatment of various cancers and infectious diseases (as reviewed in ref. 1). On the other hand, rapid progresses in genome and transcriptome analysis have given a new impetus to the field when it was realized that the antisense approach might be a strategy of choice for functional genomics and for therapeutic target validation.

New Insights Into the Membrane Translocating Process of the Tat Peptide Used for Cellular Delivery of Various Biological Molecules

A 14-mer peptide derived from the HIV Tat protein was shown to translocate efficiently through the plasma membrane [1], Basic amino acid residues in this highly cationic peptide are responsible for its internalization [2]. Non-permeant peptides, antisense oligonucleotides, proteins and even particulate material can be efficiently internalized when chemically conjugated or genetically fused to this Tat peptide. However, the mechanism of this uptake still remains to be elucidated.