Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.     

Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.     

Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners

Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues “fine tune” the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.     

Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

A multidimensional chromatographic regimen has been used to isolate and purify a peptide showing immunoreactivity for neuromedin U from guinea pig small intestine. Microsequence Edman N-terminal analysis and C-terminal analysis by enzymatic digestion showed this peptide to be a nonapeptide with the following sequence: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The C-terminal octapeptide of this sequence is the same as porcine NMU-8, and the C-terminal heptapeptide is identical to rat NMU(17-23).     

Molecular mechanism of NPF recognition by EH domains

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.     

Synthesis and photolytic cleavage of bovine insulin B22-30 on a nitrobenzoylglycyl-poly (ethylene glycol) support

The synthesis of the C-terminal nonapeptide of bovine insulin B-chain is described. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) Mr = 15,000) was used as soluble support. The C-terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.     

The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum

Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms.     

Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc,Helix aspersa,and in several other invertebrates

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 regionspecific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.     

The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency

Chemerin is a novel protein identified as the natural ligand of ChemR23 (chemerinR), a previously orphan G protein-coupled receptor expressed in immature dendritic cells and macrophages. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active, but converted into a full agonist of chemerinR by proteolytic removal of the last six amino acids. In the present work, we have synthesized a number of peptides derived from the C-terminal domain of human prochemerin and have investigated their functional properties as agonists or antagonists of human chemerinR. We found that the nonapeptide (149)YFPGQFAFS(157) (chemerin-9), corresponding to the C terminus of processed chemerin, retained most of the activity of the full-size protein, with regard to agonism toward the chemerinR. Extension of this peptide at its N terminus did not increase the activity, whereas further truncations rapidly resulted in inactive compounds. The C-terminal end of the peptide appeared crucial for its activity, as addition of a single amino acid or removal of two amino acids modified the potency by four orders of magnitude. Alanine-scanning mutagenesis identified residues Tyr(149), Phe(150), Gly(152), Phe(154), and Phe(156) as the key positions for chemerinR activation. A modified peptide (YHSFFFPGQFAFS) was synthesized and iodinated, and a radioligand binding assay was established. It was found that the ability of the various peptides to activate the chemerin receptor was strictly correlated with their affinity in the binding assay. These results confirm that a precise C-terminal processing is required for the generation of a chemerinR agonist. The possibility to restrict a medium sized protein to a nonapeptide, while keeping a low nanomolar affinity for its receptor is unusual among G protein-coupled receptors ligands. The identification of these short bioactive peptides will considerably accelerate the pharmacological analysis of chemerin-chemerinR interactions.     

Neuropeptide F and its expression in the yellow fever mosquito,Aedes aegypti

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal.     

Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family.

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.     

Synthesis of igercine--the C-terminal nonapeptide from human immunoglobulin E

Conventional methods of peptide chemistry have been used to synthesize the C-terminal nonapeptide from human immunoglobulin E, which is a potential cytophilic binding site of the IgE molecule responsible for its primary recognition and binding to specific target cell receptors.     

Evolution of neuropeptide Y and its related peptides

1. The neuropeptide Y (NPY) family of peptides includes also the gut endocrine peptide YY (PYY), tetrapod pancreatic polypeptide (PP), and fish pancreatic peptide-tyrosine (PY). All peptides are 36 amino acids long.2. Sequences from many types of vertebrates show that NPY has remained extremely well conserved throughout vertebrate evolution with 92% identity between mammals and cartilaginous fishes.3. PYY has 97–100% identity between cartilaginous fishes and bony fishes, but is less conserved in amphibians and mammals (83% identity between amphibians and sharks and 75% identity between mammals and sharks).4. NPY and PYY share 70–80% identity in most species.5. Both NPY and PYY were present in the early vertebrate ancestor because both peptides have been found in lampreys.6. The tissue distribution appears to have been largely conserved between phyla, except that PYY has more widespread neuronal expression in lower vertebrates.7. Pancreatic polypeptide has diverged considerably among tetrapods leaving only 50% identity between mammals, birdsJreptiles and frogs.8. Several lines of evidence suggest that the PP gene arose by duplication of the PYY gene, probably in the early evolution of the tetrapods.9. The pancreatic peptide PY found in anglerfish and daddy sculpin may have resulted from an independent duplication of the PYY gene.10. The relationships of the recently described mollusc and worm peptides NPF and PYF with the NPY family still appear unclear.     

Enhancement of in vitro release of FSH from rat pituitaries by a synthetic nonapeptide fragment of human seminal plasma inhibin

A synthetic C-terminal nonapeptide fragment of human seminal plasma inhibin preferentially enhances the basal release of FSH from rat pituitaries incubated in vitro, which indicates a direct action of the peptide on the pituitary. However, in the presence of LHRH, both FSH and LH release was increased particularly at higher doses of the nonapeptide. There was no change in prolactin release at 5 and 50 ng/ml but prolactin release was suppressed significantly at 500 ng/ml.     

Structure of the Eps15-stonin2 complex provides a molecular explanation for EH-domain ligand specificity

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.     

Cloning and sequencing of the bovine gastrin gene

In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed by an amidation-site (Gly-Arg-Arg), and a C-terminal nonapeptide. Comparison with human, porcine, and rat cDNA sequences revealed extensive homology in the coding region as well as in short noncoding structures.     

Enzymatic synthesis of delta sleep-inducing peptide

The delta sleep-inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain- or alpha-chymotrypsin-mediated synthesis. Secondary hydrolysis was suppressed by introducing N alpha-protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C-terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high-performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences.     

Suppression of steroidogenesis in mice granulosa cells by a synthetic nonapeptide fragment of human seminal plasma inhibin.

A synthetic nonapeptide, which is C-terminal sequence of 94-amino acid of prostatic inhibin peptide was tested for progesterone and estrogen secretion by mouse granulosa cell cultures. Nonapeptide suppressed the progesterone and estrogen synthesis, the magnitude of suppression was highest at 5 ng dose level for progesterone and 50 ng dose level for estradiol. The study suggests that, nonapeptide exerts its effect by impairing the binding of FSH to granulosa cell receptors.     

Cloning and characterization of a novel cDNA sequence encoding the precursor of a novel venom peptide (BmKbpp) related to a bradykinin-potentiating peptide from Chinese scorpion Buthus martensii Karsch

Based on the amino acid sequence of a bradykinin-potentiating peptide (Bpp) (peptide K-12) from scorpion Buthus occitanus, a full-length cDNA sequence encoding the precursor of a novel venom peptide (named BmKbpp) related to this Bpp, has been isolated and analyzed. The cDNA encodes a precursor of 72 amino acid residues, including a signal peptide of 22 residues and an extra Arg-Arg-Arg tail at the C-terminal end of the precursor, which have to be removed in the processing step. The C-terminal region (21 residues) of the precursor is homologous (57% identical) with the sequence of peptide K-12. Thus, according to the primary structure of the BmKbpp precursor, there may be a propeptide between the signal peptide and the putative mature BmKbpp at the C-terminal region of the precursor.     

Identification of novel antifungal nonapeptides through the screening of combinatorial peptide libraries based on a hexapeptide motif

Four sets of mixture based nonapeptide libraries derived from an antifungal hexapeptide pharmacophore Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH(2) (II) have been synthesized. The three C-terminal positions 7, 8 and 9 were subject to randomization using 19 genetically coded amino acids. They were then screened for their antifungal activity against Candida albicans and Cryptococcus neoformans in order to quantify inhibition at each step of the nonapeptide sublibrary deconvolution. The studies led to the identification of several novel nonapeptides with potent antifungal activity. Two of the nonapeptides exhibited approximately 17-fold increase in the activity in comparison to the lead hexapeptide motif His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH(2) (I) against C. albicans.