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Three different prohormones yield a variety of Hydra-RFamide (Arg-Phe-NH2) neuropeptides in Hydra magnipapillata.

ABSTRACT: The freshwater polyp Hydra is the most frequently used model for the study of development in cnidarians. Recently we isolated four novel Arg-Phe-NH2 (RFamide) neuropeptides, the Hydra-RFamides I-IV, from Hydra magnipapillata. Here we describe the molecular cloning of three different preprohormones from H. magnipapillata, each of which gives rise to a variety of RFamide neuropeptides. Preprohormone A contains one copy of unprocessed Hydra-RFamide I (QWLGGRFG), II (QWFNGRFG), III/IV [(KP)HLRGRFG] and two putative neuropeptide sequences (QLMSGRFG and QLMRGRFG). Preprohormone B has the same general organization as preprohormone A, but instead of unprocessed Hydra-RFamide III/IV it contains a slightly different neuropeptide sequence [(KP)HYRGRFG]. Preprohormone C contains one copy of unprocessed Hydra-RFamide I and seven additional putative neuropeptide sequences (with the common N-terminal sequence QWF/LSGRFGL). The two Hydra-RFamide II copies (in preprohormones A and B) are preceded by Thr residues, and the single Hydra-RFamide III/IV copy (in preprohormone A) is preceded by an Asn residue, confirming that cnidarians use unconventional processing signals to generate neuropeptides from their precursor proteins. Southern blot analyses suggest that preprohormones A and B are each coded for by a single gene, whereas one or possibly two closely related genes code for preprohormone C. Northern blot analyses and in situ hybridizations show that the gene coding for preprohormone A is expressed in neurons of both the head and foot regions of Hydra, whereas the genes coding for preprohormones B and C are specifically expressed in neurons of different regions of the head. All of this shows that neuropeptide biosynthesis in the primitive metazoan Hydra is already rather complex.

SUBMITTER: Darmer D

PROVIDER: S-EPMC1219495 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Project description:The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH₂. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH₂, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1' L-isoleucine (fPrI), L-cysteine (fPrC) or D-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the D-Arg residue in position P1 and thrombin are similar to those observed for the L-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 D-Arg and a bulkier P1' residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the D-stereochemistry of the residues at positions P1 and P1'.

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Three different prohormones yield a variety of Hydra-RFamide (Arg-Phe-NH2) neuropeptides in Hydra magnipapillata.

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