FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

Two alternatively spliced variants of an orphan Caenorhabditis elegans G‐protein‐coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C‐terminal tail of the receptor. A calcium mobilization assay was used as a read‐out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp‐18 precursor gene, leading to their designation as FLP‐18R1a (Y58G8A.4a) and FLP‐18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP‐18 C‐terminal signature, ‐PGVLRF‐NH 2 , were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP‐18R1 variants were fully functional at 37°C. However, a 37 to 28°C temperature shift improved their activity, an effect that was more pronounced for FLP‐18R1a. Despite differences in the C‐terminus, the region implicated in distinct G‐protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U‐73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca 2+ signal. A weak Gs‐mediated receptor activation was also detected as reflected in an agonist‐triggered concentration‐dependent cAMP increase. The matching of the FLP‐18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 339–348, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93518/1/20850_ftp.pd

Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNAPro acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar–phosphate backbone interactions in recognition of human tRNAPro. Incorporation of site-specific 2′-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2′-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving ‘direct readout’ of nucleotide bases to one relying primarily on backbone-specific ‘indirect readout’

Bis(N-methyl-N-phenyl­carbamo­yl)disulfane

The title compound, C16H16N2O2S2, has been synthesized by several different high-yield routes, and has been encountered as a co-product in a number of reaction pathways, ever since it became of inter­est to our research program over 30 years ago. We now confirm the proposed mol­ecular structure in which the mol­ecule exhibits a twofold axis of symmetry through the mid-point of the S—S bond and the two planes defined by the (carbamo­yl)sulfenyl moieties are essentially perpendicular to each other [dihedral angle = 81.55 (14)°]