Intracellular peptide delivery using amphiphilic lipid-based formulations.

Peptides, highly diverse by their nature, are important biochemical and pharmaceutical tools: ligands for cellular receptors, transcription factors, immunosuppressants, vaccines, etc. As the majority of their targets are intracellular, peptides need to cross the plasma membrane and gain access to the cytoplasm. However, due to their physicochemical properties, most peptides need to be entrapped by a molecular vehicle to be able to reach the cytosol compartment. In this study, we present new biological tools to enhance intracellular peptides delivery. Based on electrostatic interactions, two complementary types of amphiphilic molecules have been designed as delivery vehicles. A diverse set of fluorescently labeled peptides have successfully been delivered. This opens the avenue for the use of peptides combined to delivery vehicles as therapeutic aids.

Cationic lipid-mediated intracellular delivery of antibodies into live cells.

The ability to introduce antibodies to live cells opens new insights to a wide range of applications, such as protein intracellular trafficking studies, protein interference studies with blocking antibodies, and live immunolabeling or protein phosphorylation states studies. Apart from single-chain format variable (scFv) antibodies, DNA transfection of eukaryotic cells is rarely used to produce antibodies in situ, mainly due to inappropriate folding of the antibody in the cytoplasm. Thus, the development of dedicated carriers is needed since antibodies, which are large, unable to cross the plasma membrane and effective release of the antibody in the cytoplasm need to be overcome. We studied these two crucial steps using a dedicated delivery reagent in live cells and compared the results with immunocytochemistry experiments in fixed cells.

A practical approach for intracellular protein delivery.

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.

Assessing the role of the active-site cysteine ligand in the superoxide reductase from Desulfoarculus baarsii.

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of O(2)(.) to H(2)O(2), providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe(2+) center in a [His(4) Cys(1)] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu(114), is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu(114) carboxylic lateral chain affects the strength of the S-Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S-Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H(2)O(2) by a specific strengthening of the Fe-O bond. A fine tuning of the strength of the S-Fe bond by the presence of Glu(114) appears to be an essential factor for both the strength of the Fe-O bond and the pK(a) value of the Fe(3+)-peroxo intermediate species to form the reaction product H(2)O(2).

Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChE.

In a previous report, Morel and Massoulié showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulié, 2000). They reported that a 5' Bungarus fragment could partially transfer this property to a CAT expression vector. This appeared to offer the possibility of increasing the production of recombinant proteins. In the present paper, we show that insertion of this fragment in the transcribed region, before the polyadenylation site, may have either stimulatory or inhibitory effects, depending on the vector and on the reporter gene. Since the stimulatory effect of Bungarus coding region could not be attached to a small number of discrete motifs, we reasoned that it might result from a general feature of the sequence. Therefore it might be possible to partially transfer this property to the very homologous human AChE (hHAChE) coding sequence by modifications based on synonymous codons, which increased nucleotide identity between the 5' fragment (721 nucleotides) of bBAChE and hHAChE from 71% to 85%. The production of human AChE in transfected COS cells was increased nearly 2-fold with this modified construct, but still remained about 4-fold smaller than that of Bungarus AChE. There was no change in expression level in transformed Pichia pastoris. We thus confirm that coding sequences can strongly influence gene expression, but in a manner that depends on the context and cannot yet be predicted.

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism.

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.