15-Lipoxygenase-1 biosynthesis of 7S,14S-diHDHA implicates 15-lipoxygenase-2 in biosynthesis of resolvin D5

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

ABSTRACTThe oxylipins, 7S,14S-diHDHA and 7S,17S-diHDHA (RvD5), have been found in macrophages exudates and are believed to function as specialized pro-resolving mediators (SPM’s). Their biosynthesis is thought to proceed through sequential oxidations of docosahexaenoic acid (DHA) by lipoxygenase enzymes, specifically by h5-LOX first to 7S-HDHA, followed by h12-LOX to form 7S,14S-diHDHA or h15-LOX-1 to form 7S,17S-diHDHA (RvD5). In this work, we determined that oxidation of 7S-HpDHA to 7S,14S-diHDHA can be performed by either h12-LOX or h15-LOX-1, with similar kinetics. The oxidation at C14 of DHA by h12-LOX was expected, but the non-canonical reaction of h15-LOX-1 to make primarily 7S,14S-diHDHA was unexpected. Computer modeling suggests the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of I399, forcing the hydrogen abstraction from C12 to oxygenate on C14, and not C17. This result raised questions regarding synthesis of 7S,17S-diHDHA (RvD5). Strikingly, we find h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages, suggests it may have a greater role in biosynthesizing SPM’s than previously thought. We also determined that the reactions of h5-LOX with 14S-HpDHA and 17S-HpDHA are kinetically slow compared to DHA, suggesting these may be minor biosynthetic routes in-vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micro-molar potencies, which could directly regulate clot resolution.

Molecular determinants for agonist recognition and discrimination in P2X2 receptors

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.

Inter-residue interactions in alpha-helical transmembrane proteins

Motivation The number of available membrane protein structures has markedly increased in the last years and, in parallel, the reliability of the methods to detect transmembrane (TM) segments. In the present report, we characterized inter-residue interactions in α-helical membrane proteins using a dataset of 3462 TM helices from 430 proteins. This is by far the largest analysis published to date. Results Our analysis of residue–residue interactions in TM segments of membrane proteins shows that almost all interactions involve aliphatic residues and Phe. There is lack of polar–polar, polar–charged and charged–charged interactions except for those between Thr or Ser sidechains and the backbone carbonyl of aliphatic and Phe residues. The results are discussed in the context of the preferences of amino acids to be in the protein core or exposed to the lipid bilayer and to occupy specific positions along the TM segment. Comparison to datasets of β-barrel membrane proteins and of α-helical globular proteins unveils the specific patterns of interactions and residue composition characteristic of α-helical membrane proteins that are the clue to understanding their structure. Availability and implementation Results data and datasets used are available at http://lmc.uab.cat/TMalphaDB/interactions.php. Supplementary information Supplementary data are available at Bioinformatics online.

Probing the role of the backbone carbonyl interaction with the CuA center in azurin by replacing the peptide bond with an ester linkage

Replacing a backbone amide bond that is 2.17 Å from an engineered CuA center in azurin by an ester bond strengthens the Cu–Cu bond.

Synthesis, Bioassay and NMR Study of Methyleneoxy Isosters of Oxytocin and Vasopressin

Syntheses of pseudodipeptides H-Tyrψ[CH2O]Ile-OH and H-Tyrψ[CH2O]Phe-OH were carried out using the intramolecular Williamson reaction of O-benzyltyrosinol with ethyl chloroacetate followed by N-protection and aldol reaction of the corresponding morpholin-3-one in position C2 with butanone or benzaldehyde, elimination of the hydroxy group to give derivatives with a double bond either as the E/Z (1 : 1) diastereomeric mixture in the case of the former derivative or as the Z-isomer only in the case of the latter one. Stereoselective hydrogenation and hydrolysis of both the lactams yielded the corresponding pseudodipeptides lacking the carbonyl group as a hydrogen bond donor. The introduction of the pseudodipeptides into positions 2 and 3 of oxytocin and vasopressin caused total absence of all biological activities in the formed analogues. The results of the bioassay and NMR study confirmed the importance of the H-bond between the backbone carbonyl of the Tyr2 and NH proton of the Asn5 residues for stabilization of the β-turn in the cyclic hexapeptide part of both the hormones and for their biological activity.