New aspects of the structure and mode of action of the human cathelicidin LL-37 revealed by the intrinsic probe p-cyanophenylalanine.

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

Effects on antigen-presenting cells of short-term interaction with the human host defence peptide ß-defensin 2.

ß-Defensins are antimicrobial peptides that exert their host-defence functions at the interface between the host and microbial biota. They display a direct, salt- and medium-sensitive cidal activity, in vitro, against a broad spectrum of bacteria and fungi, and there is increasing evidence that they also play a role in alerting and enhancing cellular components of innate and adaptive immunity. Their interaction with biological membranes plays a central role in both of these types of activities. In the present study, we have investigated the interaction of fluorescently labelled hBD2 (human ß-defensin 2) with monocytes, macrophages and iDCs (immature dendritic cells), observing a differential capacity to be rapidly internalized into these cells. Complementary microscopy techniques [TEM (transmission electron microscopy), optical microscopy and IR microspectroscopy] were used to explore the functional and biological implications of these interactions on iDCs. Short-term exposure to the peptide resulted in significant alterations in membrane composition and re-organization of the endomembrane system, with the induction of degranulation. These events may be associated with the antigen-presenting activities or the chemotaxis of iDCs, which appears to occur via both CCR6 (CC chemokine receptor 6)-dependent and -independent mechanisms.

Artificial beta-defensin based on a minimal defensin template.

We have designed and chemically synthesized an artificial beta-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged beta-sheet core structure of natural beta-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys-->Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human beta-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.

Structure dependence of biological activities for primate cathelicidins.

We have analysed the effects of variations in orang-utan (ppy), rhesus macaque (mmu) and leaf eater (pob) monkey orthologues of the human cathelicidin LL-37, on a range of relevant biological activities. These host defence peptides range in cationicity from +4 to +10, and while the more cationic pob and mmuRL-37 are in a monomeric and unstructured form in bulk solution (F-form), the human and ppyLL-37 are in an aggregated/helical form (A-form). The in vitro antibacterial activity depended strongly on both the structural form and the charge. F-form peptides were more potent against Gram-positive and -negative bacteria and less salt, medium or serum sensitive than A-form ones. CD studies suggested that A- and F-form peptides interact with LPS in different manners, but the ability to detoxify it did not correlate directly with either the charge or structure. Toxicity towards eukaryotic cells also showed a varied dependence on the peptides' physical characteristics. Haemolytic activity was similar for all the tested peptides while other cytotoxicity assays revealed the highly cationic, F-form pobRL-37 as the most toxic, followed by the A-form human LL-37. As shown with the human peptide, toxicity depended markedly on the nature and metabolic state of the target cell. Our results suggest that different evolutionary trajectories for each orthologue lead to distinct sets of physical characteristics, which significantly differentiates their biological activities.

Primate cathelicidin orthologues display different structures and membrane interactions.

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Structuring and interactions of human beta-defensins 2 and 3 with model membranes.

beta-Defensins play an important role in both innate and adaptive immunity, displaying a direct anti-microbial activity against a wide variety of micro-organisms as well as interesting immuno-modulatory effects on host cells. Interaction with biological membranes appears to be a central theme in modulating these activities, leading to different consequences such as membrane lysis, translocation into the cytoplasm or transfer to a receptor. We have investigated the structuring of human beta-defensins (hBD2 and hBD3) and rationally designed variants, in relation to their interactions with real and model membranes. Biophysical methods, such as circular dichroism (CD), transmission or reflection IR and dye release were used to probe their structure/activity in the presence of model membranes, while fluorimetric and flow cytometric assays were used to investigate the effects on prokaryotic cells. Our results indicate that structural features, such as the helical N-terminal domains and oligomerisation at the membrane surface, may modulate the efficiency of membrane insertion and selectivity for microbial or host-cell membranes. We propose that both peptides interact with membranes as extended beta-sheet platforms that present amphipathic helices for insertion into the lipid bilayer.