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J. Biol. Chem., Vol. 281, Issue 1, 383-391, January 6, 2006

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Mechanistic and Functional Studies of the Interaction of a Proline-rich Antimicrobial Peptide with Mammalian Cells^*

Linda Tomasinsig, Barbara Skerlavaj, Niv Papo¶, Barbara Giabbai1, Yechiel Shai¶, and Margherita Zanetti2

From the Department of Biomedical Sciences and Technology, University of Udine, P.le Kolbe 4, I-33100 Udine, Italy, National Laboratory Interuniversity Consortium for Biotechnology, Area Science Park, Padriciano 99, I-34012 Trieste, Italy, and ^¶Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

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.

Received for publication, September 20, 2005 , and in revised form, October 26, 2005.

^* This work was supported by grants from the Italian Ministry for University and Research (P.R.I.N), the Interuniversity Consortium for Biotechnology, and Regione FVG. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Biocrystallography Unit, Dept. of Biological and Technological Research, San Raffaele Scientific Institute, I-20132 Milan, Italy.

² To whom correspondence should be addressed: Dept. of Biomedical Sciences and Technology, University of Udine, P.le Kolbe 4, I-33100 Udine, Italy. Tel.: 39-0432-494390; Fax: 39-0432-494301; E-mail: zanetti{at}icgeb.trieste.it.

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