Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.

BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands.

BACKGROUND: Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. METHODS: Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. RESULTS: Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. CONCLUSIONS: AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. GENERAL SIGNIFICANCE: Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system.

Polymer-Supported Optically Active fac(S)-Tris(thiotato)rhodium(III) Complex for Sulfur-Bridging Reaction With Precious Metal Ions.

The optically active mixed-ligand fac(S)-tris(thiolato)rhodium(III) complexes, ΔL -fac(S)-[Rh(aet)2 (L-cys-N,S)](-) (aet = 2-aminoethanethiolate, L-cys = L-cysteinate) () and ΔLL -fac(S)-[Rh(aet)(L-cys-N,S)2 ](2-) were newly prepared by the equatorial preference of the carboxyl group in the coordinated L-cys ligand. The amide formation reaction of with 1,10-diaminodecane and polyallylamine gave the diamine-bridged dinuclear Rh(III) complex and the single-chain polymer-supported Rh(III) complex with retention of the ΔL configuration of , respectively. These Rh(III) complexes reacted with Co(III) or Co(II) to give the linear-type trinuclear structure with the S-bridged Co(III) center and the two Δ-Rh(III) terminal moieties. The polymer-supported Rh(III) complex was applied not only to the CD spectropolarimetric detection and determination of a trace of precious metal ions such as Au(III), Pt(II), and Pd(II) but also to concentration and extraction of these metal ions into the solid polymer phase. Chirality 28:85-91, 2016. © 2015 Wiley Periodicals, Inc.

Identification of Novel Single-Domain Antibodies against FGF7 Using Phage Display Technology.

Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor (FGF) family of proteins. FGF7 is of stromal origin and produces a paracrine effect on epithelial cells. In the current investigation, we aimed to identify new single-domain antibodies (sdAbs) against FGF7 using phage display technology. The vector harboring the codon-optimized DNA sequence for FGF7 protein was transformed into Escherichia coli BL21 (DE3) pLysS, and then the protein was expressed at the optimized condition. Enzyme-linked immunosorbent assay, circular dichroism spectropolarimetry, and in vitro scratch assay experiments were used to confirm the proper folding and functionality of the purified FGF7 protein. The purity of the produced FGF7 was 92%, with production yield of 3.5 mg/L of culture. Panning against the purified FGF7 was performed, and the identified single-domain antibodies showed significant affinity. Further investigation on one of the selected sdAb displaying phage clones showed concentration-dependent binding to FGF7. The selected sdAb can be used for developing novel tumor-suppressing agents where inhibition of FGF7 is required.