A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.     

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.     

Conserved Chloroplast Open-reading Frame ycf54 Is Required for Activity of the Magnesium Protoporphyrin Monomethylester Oxidative Cyclase in Synechocystis PCC 6803

The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.     

Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis

An in-depth understanding of the molecular and cellular complexity of angiogenesis continues to advance as new stimulators and inhibitors of blood vessel formation are uncovered. Gaining a more complete understanding of the response of blood vessels to both stimulatory and inhibitory molecules will likely contribute to more effective strategies to control pathological angiogenesis. Here, we provide evidence that endothelial cell interactions with structurally altered collagen type IV may suppress the expression of insulin-like growth factor binding protein-4 (IGFBP-4), a well documented inhibitor of the IGF-1/IGF-1R signaling axis. We report for the first time that IGFBP-4 differentially inhibits angiogenesis induced by distinct growth factor signaling pathways as IGFBP-4 inhibited FGF-2- and IGF-1-stimulated angiogenesis but failed to inhibit VEGF-induced angiogenesis. The resistance of VEGF-stimulated angiogenesis to IGFBP-4 inhibition appears to depend on sustained activation of p38 MAPK as blocking its activity restored the anti-angiogenic effects of IGFBP-4 on VEGF-induced blood vessel growth in vivo. These novel findings provide new insight into how blood vessels respond to endogenous inhibitors during angiogenesis stimulated by distinct growth factor signaling pathways.     

Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.     

Structures of peptide agonists for human protease activated receptor 2

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor in being self-activated, after pruning of the N-terminus by serine proteases like trypsin and tryptase. Short synthetic peptides corresponding to the newly exposed N-terminal hexapeptide sequence also activate PAR2 on immunoinflammatory, cancer and many normal cell types. (1)H nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy were used here to search for structural clues to activating mechanisms of the hexapeptide agonists SLIGRL (rat), SLIGKV (human) and the peptidomimetic analogue, 2-furoyl-LIGRLO. Either with a free or acetyl capped N-terminus, these agonist peptides display significant propensity in aprotic (DMSO) or lipidic (water-SDS) solvents for turn-like conformations, which are predicted to be receptor-binding conformations in the transmembrane or loops region of PAR2. These motifs may be valuable for the design of small molecule PAR2 agonists and antagonists as prospective new drugs for regulating inflammatory and proliferative diseases.     

Identification, optimisation and in vivo evaluation of oxadiazole DGAT-1 inhibitors for the treatment of obesity and diabetes

A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening (HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid containing clinical candidate 53 (AZD3988), which demonstrated excellent DGAT-1 potency (0.6 nM), good pharmacokinetics and pre-clinical in vivo efficacy that could be rationalised through a PK/PD relationship.