Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells.
Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.
Tomasinsig
Linda
L
Department of Biomedical Sciences and Technology, University of Udine, P.le Kolbe 4, I-33100 Udine, Italy.
Skerlavaj
Barbara
B
Papo
Niv
N
Giabbai
Barbara
B
Shai
Yechiel
Y
Zanetti
Margherita
M
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000493:pharmacokinetics*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004705:Endocytosis; D004926:Escherichia coli / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008322:Mammals; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D010713:Phosphatidylcholines / Q000378:metabolism; D010873:Pinocytosis; D011392:Proline / Q000378:metabolism; D016196:S Phase; D017550:Spectroscopy, Fourier Transform Infrared; D013211:Staphylococcus aureus / Q000187:drug effects; D020349:Surface Plasmon Resonance; D020298:U937 Cells
D023181:Antimicrobial Cationic Peptides; C481640:Bac7(1-35) peptide; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D004247:DNA; D002784:Cholesterol; D011392:Proline