Determination of Membrane Immersion Depth with O2: A High-Pressure 19F NMR Study

AbstractOxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (PO2) of 100Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depth-dependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF2)5C2H4-O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates (R1P) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by 19F NMR. The paramagnetic relaxation rates, R1P, of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R1P to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed

Interaction and Cellular Localization of the Human Host Defense

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response. Cationic host defense peptides are key components of innate immunity that have both direct, broad-spectrum antimicrobial activity and an ability to stimulate immunity against bacteria, fungi, parasites, and viruses (14). In evolutionary terms, th

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide*

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases