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

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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 is the third helix of the homeodomain of Antennapedia protein, known to internalize into cells, partly via receptor-independent process, since its reverso, enantio, and retro-inverso peptide analogues are also internalized as cell-penetrating peptides (CPPs). The ability of Penetratin to cause non-cytotoxic cell membrane perturbation now appears as one way to explain its passage across the plasma membrane to reach the cytosol of cells (or internalization), a process in which membrane lipids are key actors. Penetratin also translocates into model membranes (LUV, GUV) and plasma membrane vesicles. Whatever the mechanism of membrane translocation is, lipids are initial contact points for the peptide. Photoactivatable phospholipids have been widely used to study biological systems. The potential of benzophenone for the efficient photolabeling in peptide/lipid and lipid/lipid complexes is well established. 

 

 

Altogether these results are in good agreement with a previous study on the interaction of Penetratin with binary lipid mixtures that showed that the peptide always recruits the lipid with the lower phase transition. Our data establish that Penetratin interacts preferentially with phospholipids found in disordered membrane domains. Whether this is also the case in biological membranes remains to be fully demonstrated. Therefore, we hope this method to be useful for further applications in biological systems, to study insertion depth of membranotropic peptides in general, and to delineate lipid preference of other well-known cell-penetrating peptides, such as Tat, in relation with their membrane translocation properties. 

 

 

Résumé: 

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.

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

Chen-Yu Jiao, Emmanuelle Sachon, Isabel D. Alves, Gérard Chassaing, Gérard Bolbach and Sandrine Sagan

Angew Chem Int Ed. 2017 May 9

doi: 10.1002/anie.201703465