Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.     

Ephrin‐A5 regulates the formation of the ascending midbrain dopaminergic pathways

Dopaminergic neurons from the substantia nigra and the ventral tegmental area of the midbrain project to the caudate/putamen and nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. However, the mechanisms underlying the development of these pathways are not well understood. In the current study, the EphA5 receptor and its corresponding ligand, ephrin‐A5, were shown to regulate dopaminergic axon outgrowth and influence the formation of the midbrain dopaminergic pathways. Using a strain of mutant mice in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, EphA5 protein expression was detected in both the ventral tegmental area and the substantia nigra of the midbrain. Ephrin‐A5 was found in both the dorsolateral and the ventromedial regions of the striatum, suggesting a role in mediating dopaminergic axon‐target interactions. In the presence of ephrin‐A5, dopaminergic neurons extended longer neurites in in vitro coculture assays. Furthermore, in mice lacking ephrin‐A5, retrograde tracing studies revealed that fewer neurons sent axons to the striatum. These observations indicate that the interactions between ephrin‐A ligands and EphA receptors promote growth and targeting of the midbrain dopaminergic axons to the striatum. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Intracellular K+ sensing of SKOR, a Shaker-type K+ channel from Arabidopsis

Most K+ channels in plants are structurally classified into the Shaker family named after the shaker K+ channel in Drosophila. Plant K+ channels function in many physiological processes including osmotic regulation and K+ nutrition. An outwardly rectifying K+ channel, SKOR, mediates the delivery of K+ from stelar cells to the xylem in the roots, a critical step in the long-distance distribution of K+ from roots to the upper parts of the plant. Here we report that SKOR channel activity is strictly dependent on intracellular K+ concentrations. Activation by K+ did not affect the kinetics of voltage dependence in SKOR, indicating that a voltage-independent gating mechanism underlies the K+ sensing process. Further analysis showed that the C-terminal non-transmembrane region of the SKOR protein was required for this sensing process. The intracellular K+ sensing mechanism couples SKOR activity to K+ nutrition status in the 'source cells', thereby establishing a supply-based unloading system for the regulation of K+ distribution.     

Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 �� Glucanase Gene During Compatible Plant-Nematode Interactions

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5’ and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between −1,673 and −1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.     

Axoplasm isolation from peripheral nerve

Localized changes in the composition of axonal cytoplasm (axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon‐glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon‐enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Global and local mechanisms sustain axonal proteostasis of transmembrane proteins

The control of neuronal protein homeostasis or proteostasis is tightly regulated both spatially and temporally, assuring accurate and integrated responses to external or intrinsic stimuli. Local or autonomous responses in dendritic and axonal compartments are crucial to sustain function during development, physiology and in response to damage or disease. Axons are responsible for generating and propagating electrical impulses in neurons, and the establishment and maintenance of their molecular composition are subject to extreme constraints exerted by length and size. Proteins that require the secretory pathway, such as receptors, transporters, ion channels or cell adhesion molecules, are fundamental for axonal function, but whether axons regulate their abundance autonomously and how they achieve this is not clear. Evidence supports the role of three complementary mechanisms to maintain proteostasis of these axonal proteins, namely vesicular transport, local translation and trafficking and transfer from supporting cells. Here, we review these mechanisms, their molecular machineries and contribution to neuronal function. We also examine the signaling pathways involved in local translation and their role during development and nerve injury. We discuss the relative contributions of a transport‐controlled proteome directed by the soma (global regulation) versus a local‐controlled proteome based on local translation or cell transfer (local regulation).      

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase‐3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase‐3 activity using an in ovo electroporation method. The axon arborization pattern was 3‐dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6–8, there appeared to be a dynamic balance in the axon arborization pattern between the “targeting” mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the “pathfinding” mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.