Self-aggregation of spin-labeled alamethicin in ePC vesicles studied by pulsed electron-electron double resonance

The pulsed electron-electron double resonance technique was used to study the dipole-dipole interactions between 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels located at position 16 of an alamethicin analogue in ePC vesicles that were frozen to 77 K. We show that under these conditions the alamethicin molecules tend to form aggregates over the range of peptide concentrations 3.5 x 10(-3) to 1 x 10(-2) M. The number of molecules in the aggregate is found to be 4.2 +/- 0.2. A spin-label distance distribution function is also obtained with a maximum at a distance of 2.3 nm and a half-height width of 1.3 nm. We envisage that these data will permit us to generate a molecular model of cellular ion channel

PELDOR Conformational Analysis of bis-Labeled Alamethicin Aggregated in Phospholipid Vesicles

Alamethicin (Alm) is a linear peptide antibiotic of great interest for its capability to form self-assembled ion channels in lipid membranes. Here, the pulsed electron electron double resonance technique was used to obtain unique conformational information on the aggregated peptide in the lipid membrane-bound state. Since a specific helical conformation implies a given length to the peptide molecule, a distance r was measured at the nanometer scale via the electron dipole-dipole interaction between two 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels synthetically incorporated at positions 1 and 16 of this 19-mer peptide. Two data sets were collected (at 77 K): (i) from aggregates of Alm in hydrated egg-yolk phosphocholine (ePC) vesicles (at peptide-to-lipid ratios of 1:200 and 1:75) and (ii) from nonaggregated Alm in pure (nonhydrated) ePC and in solvents of different polarity. The intramolecular distance between the two labels obtained in this manner is in excellent agreement with that calculated on the basis of an almost fully developed a-helical conformation for this peptide and is found to be independent of the molecular aggregated state and the environment polarity as well

Aggregation of spin-labeled alamethicin in low-polarity solutions as studied by PELDOR spectroscopy

Alamethicin is a 20-residue antibiotic peptide. Interest in studying alamethicin stems from its ability to form conducting channels in biological membranes, thus changing their permeability. Although such channels in artificial and biological membranes have been well documented (see, e.g., [1, 2] and references therein), available reliable data are still insufficient to elucidate the mechanism of action of alamethicin on the properties of membranes. Therefore, it is of interest to study the self-aggregation of alamethicin molecules in media mimicking the membrane surface and interfacial layer, for example, in polar and nonpolar media. One approach to studying peptide self-aggregation in solutions is the spin label method in combination with CW ESR and pulsed ESR electron\u2013electron double resonance (PELDOR). In this work, using these methods, we obtained the first reliable structural data on alamethicin aggregates in nonpolar media

ICOS Deficiency Results in Exacerbated IL-17 Mediated Experimental Autoimmune Encephalomyelitis

Inducible costimulatory molecule (ICOS) is important for the effector function of T cells, especially for Th2 and T cell dependent B cell responses. However, it has been shown that ICOS is required for the differentiation of Th17 cells. Since IL-17 has been identified as a major cytokine involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the enhanced severity of EAE in ICOS-deficient mice (ICOS-/-) mice is unexpected. To better understand the role of ICOS and of IL-17 in EAE, we induced EAE in ICOS-/- by immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) in complete Freund's adjuvant. As previously reported, we found that ICOS-/- mice developed more severe EAE. Upon restimulation with MOG(35-55,) splenocytes from ICOS-/- mice with EAE produced higher amounts of IL-17 and ICOS-/- mice had a higher expression of IL-17, IL-6, and TGF-beta mRNA in the spinal cords at the onset of the disease. Finally, the blockade of IL-17 strongly inhibited disease even in ICOS-/- mice, showing that IL-17 is playing a major role in the pathogenesis of EAE both in WT and ICOS-/- mice. In conclusion, MOG immunization induces MOG-specific Th17 cells also in ICOS-/- mice, and a higher expression of IL-17 and of Th17-driving cytokines IL-6 and TGF-beta in the central nervous system at the onset of EAE that correlates with their more severe disease

Averting inflammation by targeting the cytokine environment.

Cytokines are key instigators and regulators of immune responses and therefore hold great potential as targets for new therapeutic strategies. However, the selection of which cytokines to target, and in particular the identification of which cytokines regulate the rate-limiting steps of disease pathways, is crucial to the success of such strategies. Moreover, balancing the need for ablating pathological inflammatory responses and simultaneously maintaining the ability to control infectious agents is a key consideration. Recent advances in our understanding of cytokine networks, as well as technical progress in blocking cytokines in vivo, are likely to be a source for new drugs that can control chronic inflammatory diseases