Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects.
We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 23-50 (mPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced approximately 60% haemoglobin leakage after 30 min, whereas mPrPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be approximately 12% for 50 microM mPrPp(1-28), and approximately 1% for 50 microM mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.
Oglecka
Kamila
K
Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden.
Lundberg
Pontus
P
Magzoub
Mazin
M
Göran Eriksson
L E
LE
Langel
Ulo
U
Gräslund
Astrid
A
D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D005452:Fluoresceins / Q000378:metabolism; D006367:HeLa Cells; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D000072002:Prion Proteins; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D053835:Unilamellar Liposomes / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D005452:Fluoresceins; D006454:Hemoglobins; D019913:Nuclear Localization Signals; D010455:Peptides; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D053835:Unilamellar Liposomes; D014867:Water; C007740:fluorexon