Printer-friendly version

Exploiting Benzophenone Photoreactivity to Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes

Angew Chem Int Ed. 2017 May 9

 

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles depending on their phospholipid content. To evaluate the phospholipid preference of Penetratin, as the first step of translocation, we have exploited the benzophenone triplet kinetics of hydrogen abstraction, slower for secondary than for allylic hydrogens. Using multilamellar vesicles (MLVs) of various phospholipid content, we have identified and characterized the crosslinked products by MALDI-TOF mass spectrometry. Penetratin shows a preference for negatively charged (vs zwitterionic) polar heads, for unsaturated (vs saturated) and short (vs long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.

Interview Geoffrey BODENHAUSEN - Comprendre la Résonance magnétique nucléaire

La résonance magnétique nucléaire (RMN) est un outil analytique universel de détermination structurale et dynamique de la matière au sens large : produits naturels, substances synthétiques, macromolécules biologiques… jusqu’au corps humain tout entier grâce à l’imagerie par résonance magnétique (IRM). 

A cell penetrant manganese SOD-mimic is able to complement MnSOD and exerts an anti-inflammatory effect on cellular and animal models of inflammatory bowel diseases

Inorg. Chem.201756 (5), pp 2545–2555

 

Inorganic complexes are increasingly used for biological and medicinal applications and the question of the cell-penetration and of the cell-distribution of metallodrugs is key to understand their biological activity. Oxidative stress is known to be involved in inflammation and in Inflammatory Bowel Diseases for which antioxidative defenses are weakened. We report here the study of a Mn-complex Mn1 mimicking superoxide dismutase, a protein involved in the cell protection against oxidative stress, using an approach in inorganic cellular chemistry combining investigation of Mn1 intracellular speciation using mass spectrometry, of its quantification and distribution using electron paramagnetic resonance and spatially-resolved X-ray fluorescence with evaluation of its biological activity. More precisely, we have looked for and find the MS-signature of Mn1 in cell lysates and quantified the overall Mn-content. Intestinal epithelial cells activated by bacterial lipopolysaccharide were taken as a cellular model of oxidative stress and inflammation. Mn1 exerts an intracellular anti-inflammatory activity, remains at least partially coordinated, with a diffuse distribution over the whole cell and functionally complements mitochondrial MnSOD.

 

Transportable hyperpolarized metabolites

Nature Communications 8, Article number: 13975 (2017)

 

Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically o1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude.

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

Scientific Reports 6, 36938 (2016)

 

The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.