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We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016.

Estrogen-related receptor γ (ERRγ) is a constitutively active nuclear receptor functioning as a transcription factor. ERRγ binds to a single half site designated as ERRE that has only a single DNA-binding motif. However, with regard to the subunit structure, it remains a matter of controversy whether ERRγ binds as a monomer or dimer. Because the ligand-binding domain (LBD) of ERRγ was in a homodimer form in its X-ray crystal structure, the peptide fragments present in the dimer interfaces would perturb or destabilize the dimer structure by inhibiting the mutual interaction among ERRγ molecules. Thus, to demonstrate the essential homodimer structure of ERRγ, we utilized the peptides corresponding to the α-helix peptides 7 (H7), H9, and H10/11 in order to test such inhibitor activity. These selections were done based on a structural analysis of the X-ray crystal structures of ERRγ-LBD, which forms a head-to-head dimer structure. Peptides were evaluated by means of a luciferase reporter gene assay, in which ERRγ exhibited a high constitutive activity with no ligand. When the peptide was expressed in the HeLa cells together with ERRγ, these peptides clearly showed a concentration-dependent activity inhibition, indicating that ERRγ is indeed homodimerized as required for DNA transcription activity. The present results strongly suggest that human nuclear receptor ERRγ functions as a genuine homomeric dimer with symmetrical dimeric interface regions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 547-554, 2016.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean β-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%–94%) and similarity (89%–100%) to insect PDFs and also to the crustacean β-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.