NMR solution structure of human cannabinoid receptor-1 helix 7/8 peptide: Candidate electrostatic interactions and microdomain formation

We report the NMR solution structure of a synthetic 40-mer (T³⁷⁷-E⁴¹⁶) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H₂O. Structural features include, from the peptide’s amino terminus, a hydrophobic α-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic α-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide’s loop-like region. Potential cation-π and cation-phenolic OH interactions between Y³⁹⁷ in the TMH7 NPxxY motif and R⁴⁰⁵ in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1 conformer transitions.     

The influence of N-, S-containing chinasolone derivatives (NC-224) on the biochemical and physicochemical parameters of membrane endoplasmatic reticulum and nuclear chromatine fractions of rats liver cells in conditions of its injury by tetrachloromethane

Using biochemical and physicochemical methods of investigation in vivo, the effect of the substance NC-224, N-, S-chinasolone-derivative, on the lipoperoxidation activity in rat liver endoplasmatic reticulum membranes and nuclear chromatin fractions under tetrachloromethane intoxication have been studied. It was shown that NC-224 has pronounced antioxidant activity which is the biochemical basis of the substance membrane- and genome-protective effects and its ability to restore physicochemical properties of the surface and hydrophobic zones of hepatocyte membranes and structural parameter nuclear chromatin fractions in the conditions of chemical liver injury.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Interaction of fibrin(ogen) with apolipoprotein(a): further characterization and identification of a novel lysine-dependent apolipoprotein(a)-binding site within the gamma chain 287-411 region

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.     

Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin

Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid vesicles. To test the ability of HF-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of the diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used F?rster resonance energy transfer to follow the effect of HF-TAC on aggregation of aqueous and membrane-bound T-domain. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of the aqueous T-domain at low pH. The presence of HF-TAC in the buffer is demonstrated to suppress aggregation in solution and ensure correct insertion and folding of the T-domain into the lipid vesicles, without solubilizing the latter. Thus, hemifluorinated surfactants stabilize the low-pH conformation of the T-domain as a water-soluble monomer while acting as low-molecular weight chaperones for its insertion into preformed lipid bilayers.     

Is it possible to predict amyloidogenic regions from sequence alone?

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.     

A modular environmental and economic assessment applied to the production of Hermetia illucens larvae as a protein source for food and feed

The International Journal of Life Cycle Assessment - The inclusion of insect protein into the food system has been proposed as a promising solution to ensure future food security and mitigate...     

Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine phosphatase 1B

Сalix[4]arenes bearing methylenebisphosphonic or hydroxymethylenebisphosphonic acid fragments at the wide rim of the macrocycle were studied as inhibitors of PTP1B. Some of the inhibitors showed IC₅₀ values in the micromolar range and good selectivity in comparison with other protein tyrosine phosphatases such as TC-PTP, PTPβ, LAR, and CD45. Kinetic studies indicated that the calix[4]arene derivatives influence PTP1B activity as slow-binding inhibitors. Based on molecular docking results, the binding modes of the macrocyclic bisphosphonates in the active centre of PTP1B are discussed.     

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Redox regulation of the mitochondrial KATP channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK_ATP) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK_ATP regulation. mK_ATP activity was measured by both osmotic swelling and Tl⁺ flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK_ATP in cardioprotection by nitroxyl. Key findings were as follows: (i) mK_ATP was activated by O₂⁻ and H₂O₂ but not other peroxides. (ii) mK_ATP was inhibited by NADPH. (iii) mK_ATP was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK_ATP-dependent manner. Overall, these results suggest that the mK_ATP channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK_ATP may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.