The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 ؎ 0.4 and 1.8 ؎ 0.3 and K d values of 39 ؎ 18 and 103 ؎ 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane. Lactoferrin (Lf)1 is an 80-kDa iron-binding glycoprotein found in various biological secretions, mainly in milk (1), and in polymorphonuclear leukocytes (2). The biological roles of Lf include antibacterial activities through mechanisms not yet clearly elucidated. The ability of Lf to tightly chelate two ferric ions allows the protein to limit the iron availability to bacteria and ultimately causes bacteriostasis (3, 4). Moreover, Lf and an Lf-derived peptide called lactoferricin (Lfcin) (5) (residues 1-47 and 18 -41 for human (hLf) and bovine (bLf) Lfs, respectively) were shown to bind to Gram-negative bacteria, including Escherichia coli and to release lipopolysaccharides (LPS) from the outer membrane (6 -8). Stable complexes are formed between free LPS and Lf (9 -12), but the mechanism of membrane destabilization is not clearly elucidated. During the last few years, Naidu and co-workers (13-15) reported potential interactions of either hLf or bLf with porins, the major pore-forming proteins of the outer membrane of various Gram-negative bacteria. In particular, the binding of Lf to porins OmpF and OmpC in E. coli was reported (13). However, the biochemical evidence of interactions between Lf and porins was mainly based upon Western blot analyses using SDS-extracted and heat-denatured porin monomers. Because porins are potential receptors for b...

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

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