TAILIEUCHUNG - Báo cáo khóa học: Membrane interaction and cellular internalization of penetratin peptides

Penetratin is a 16-residue peptide [RQIKIWFQNRRM KWKK(43–58)]derived from the Antennapedia homeo-domain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel themembrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg resi-dues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these resi-dues to Phe. . | Eur. J. Biochem. 271 1187-1197 2004 FEBS 2004 doi Membrane interaction and cellular internalization of penetratin peptides Bart Christiaens1 Johan Grooten2 Michael Reusens1 Alain Joliot4 Marc Goethals1 3. Joel Vandekerckhove1 3 Alain Prochiantz4 and Maryvonne Rosseneu1 1 Department of Biochemistry of the Ghent University department of Molecular Biology and 3Department of Medical Protein Research of the Flanders Interuniversity Institute for Biotechnology Ghent Belgium 4Ecole Normale Superieure Paris France Penetratin is a 16-residue peptide RQIKIWFQNRRM KWKK 43-58 drrived I omm the AnleniKipedia Oomoo-domain which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and or Arg residues to Ala while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation while circular dichroism indicated that lipid binding increased the a-helical structure of the peptides. The R53A K57A and R52A K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A K57A R52A K55A and K46A K57A variants by MDCK cells was similar .

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