Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites

Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.     

A free terminal carboxylate group is required for PhrA pentapeptide inhibition of RapA phosphatase

In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators. The RapA phosphatase is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity. PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide. Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid. A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1:1 ratio and it is approximately 200 fold more active than a C-terminal amide peptide. Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA.     

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1-4 via spacer to alpha-helical NPY 25-36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1-4 linked via epsilon-aminocaproic acid (Aca) to the C-terminal partially alpha-helical peptide amide segment 25-36. NPY 1-4-Aca-25-36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain.

Subtypes of the neuropeptide Y (NPY) receptor in the rat brain were identified by the use of the selective Y-1 analog, [Leu34-Pro34] NPY. In rat brain homogenate binding studies, [Leu31-Pro34] NPY was found to produce a partial inhibition of 100 pM 125I-labeled peptide YY (PYY) binding with a plateau at 50-1000 nM [Leu31-Pro34] NPY resulting in a 70% inhibition of binding. The C-terminal fragment NPY 13-36, a putative Y-2 agonist, exhibited very little selectivity in rat brain homogenates. Scatchard analysis of 125I-labeled PYY binding to rat brain homogenate yielded biphasic plots with Kd values of 40 and 610 pM. Inclusion of 100 nM [Leu31-Pro34] NPY was found to eliminate the low affinity component of 125I-labeled PYY binding leaving a single, high affinity binding site with a Kd of 68 pM. In autoradiographic studies, displacement curves indicated that [Leu31-Pro34] NPY completely inhibited binding in the cerebral cortex with little effect on the binding in the hypothalamus. On the other hand NPY 13-36 inhibited binding in the hypothalamus at low concentrations but required higher concentrations to inhibit binding in the cerebral cortex. Other brain regions such as the hippocampus, appeared to contain both subtypes. Subsequent to these studies, a quantitative autoradiographic map was conducted using 50-100 pM 125I-labeled PYY in the presence and absence of [Leu31-Pro34] NPY which produced a selective displacement of binding in certain distinct brain regions. These areas included the cerebral cortex, certain thalamic nuclei and brainstem while ligand binding was retained in other brain regions including the zona lateralis of the substantia nigra, lateral septum, nucleus of the solitary tract and the hippocampus. Numerous brain regions appeared to contain both receptor subtypes. Therefore, the Y-1 and Y-2 receptor subtypes exhibited a somewhat distinct distribution in the brain. In addition, 125I-labeled PYY appears to label the Y-2 receptor with relatively higher affinity when compared to the Y-1 receptor.     

Conformational Analysis of Neuropeptide Y Segments by CD,NMR Spectroscopy and Restrained Molecular Dynamics

Neuropeptide Y (NPY), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for affinity to the NPY receptor subtypes Y₁ and Y₂, in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18–36) and NPY(13–36), are described. The results obtained by CD measurements, two-dimensional NMR spectros copy and a conformational refinement of the NMR-derived structure by molecular mechanics simulations support the findings of previously published structure –activity relationship studies for biologically active and selective compounds. In particular, the α-helical conformation as well as an appropriate exposure of the side chains of the critical C-terminal dipeptide within NPY(18–36) are in agreement with the prerequisites proposed for Y₂ receptor binding of that segment.     

Opiate receptor binding affinities of some D-amino acid substituted beta-casomorphin analogs

beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.     

Design of the Y1-receptor-selective cyclic peptide based on the C-terminal sequence of neuropeptide Y.

We designed four cyclic peptides which are mimics of the C-terminal region of human neuropeptide Y (NPY) on the basis of the structural model of NPY. One of these cyclic peptides, c[D-Cys29-L-Cys34]NPY Ac-29-36 (YM-42454), exhibited significantly higher affinity for the Y1-receptor than the corresponding C-terminal linear fragment, NPY Ac-28-36. Interestingly, YM-42454 showed binding affinity for the Y1-receptor in spite of the lack of the N-terminal sequence of NPY, whereas it did not show any binding affinity for the Y2-receptor. This conformationally restricted Y1-selective peptide would provide some insights into the bioactive conformation of the C-terminal region of NPY.     

Sequential processing reactions in the formation of hormone amides

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D-Tyr-Val-Gly and D-Tyr-Val-Gly-Lys-Arg to the dipeptide amide D-Tyr-Val-CONH2. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D-Tyr-Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes. The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C-terminal glycine are reactions that take place consecutively. These prohormone-processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.     

High affinity displacement of [(3)H]NPY binding to the crude venom of conus anemone by insect neuropeptides.

The venom from Conus anemone contains a protein, named ANPY toxin, which displayed high affinity (IC(50) in nanomolar range) to neuropeptide Y (NPY), [Leu(31), Pro(34)]NPY, peptide YY, pancreatic polypeptide, the Y(1) antagonist 1229U91, and C-terminal NPY fragments. N-terminal fragments and the free acid form of NPY did not bind to ANPY. The truncated NPY fragments displayed very low affinity to Y(1) receptors and partially inhibited [(3)H]NPY binding to anti-NPY antiserum. Several insect neuropeptides, the sequences of which related to the C-terminal fragments of NPY, were observed to bind with similar affinity or even 20 times higher (Lom-MS and Scg-NPF) affinity than NPY. In contrast, no significant binding of these insect peptides was observed for Y(1) receptors and anti-NPY antiserum. Therefore, ANPY can be viewed as an acceptor that binds with very high affinity to a broad spectrum of vertebrate and invertebrate neuropeptides that share a similar C-terminal amino acid sequence.     

Alpha-helical small molecular size analogues of neuropeptide Y: structure-activity relationships.

C-terminal analogues of neuropeptide Y (NPY) of small molecular size have been synthesized. The influence of chain length, single or multiple amino acid substitution, and segment substitutions on receptor binding, pre- and postsynaptic biological activity, and conformational properties have been investigated. Receptor binding and in vivo assays revealed biological activity for NPY Ac-25-36 that increased with increasing alpha-helicity. In attempts to stabilize the alpha-helical content, three independent types of modified NPY Ac-25-36 analogues were synthesized. Strong agonistic activities could be detected in a series of discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was NPY 1-4-Aca-25-36 (Aca, epsilon-aminocaproic acid). For the first time conformational properties of a series of small NPY analogues have been investigated by CD, and correlated with biological activity and receptor binding. A C-terminal dodecapeptide segment of NPY with an amount of 50% substitution to the native C-terminal sequence of NPY was found to exhibit significant receptor binding.     

Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus

Eighteen peptides were isolated from brain extracts of the stick insect Carausius morosus. The peptides were purified in four steps by high-performance liquid chromatography, monitored by their ability to inhibit juvenile hormone biosynthesis by corpora allata of the cricket Gryllus bimaculatus in vitro, and chemically characterised by Edman degradation and mass spectrometry. We obtained complete primary-structure information for nine peptides, four of which belong to the peptide family characterised by a common C-terminal pentapeptide sequence -YXFGLamide. The remaining five belong to the W(2)W(9)amide peptide family, nonapeptides characterised by having the amino acid tryptophan in positions 2 and 9. The amino-acid sequence of two other peptides could not be completely resolved by means of Edman degradation; however, these peptides could be allocated to the -YXFGLamide and the W(2)W(9)amide family, respectively, by comparison of retention times, co-elution and mass spectrometry. Both classes of neuropeptides strongly inhibit juvenile hormone biosynthesis in crickets but show no inhibiting effect on the corpora allata of the stick insect.     

Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection

The biological importance of the neuropeptide Y (NPY) has steered a number of investigations about its solution structure over the last 20 years. Here, we focus on the comparison of the structure and dynamics of NPY free in solution to when bound to a membrane mimetic, dodecylphosphocholine (DPC) micelles, as studied by 2D (1)H NMR spectroscopy. Both, free in solution and in the micelle-bound form, the N-terminal segment (Tyr1-Glu15) is shown to extend like a flexible tail in solution. This is not compatible with the PP-fold model for NPY that postulates backfolding of the flexible N terminus onto the C-terminal helix. The correlation time (tau(c)) of NPY in aqueous solution, 5.5 (+/-1.0) ns at 32 degrees C, is only consistent with its existence in a dimeric form. Exchange contributions especially enhancing transverse relaxation rates (R(2)) of residues located on one side of the C-terminal helix of the molecule are supposed to originate from dimerization of the NPY molecule. The dimerization interface was directly probed by looking at (15)N-labeled NPY/spin-labeled [TOAC34]-[(14)N]-NPY heterodimers and revealed both parallel and anti-parallel alignment of the helices. The NMR-derived three-dimensional structure of micelle-bound NPY at 37 degrees C and pH 6.0 is similar but not identical to that free in solution. The final set of 17 lowest-energy DYANA structures is particularly well defined in the region of residues 21-31, with a mean pairwise RMSD of 0.23 A for the backbone heavy atoms and 0.85 A for all heavy atoms. The combination of NMR relaxation data and CD measurements clearly demonstrates that the alpha-helical region Ala18-Thr32 is more stable, and the C-terminal tetrapeptide becomes structured only in the presence of the phosphocholine micelles. The position of NPY relative to the DPC micelle surface was probed by adding micelle integrating spin labels. Together with information from (1)H,(2)H exchange rates, we conclude that the interaction of NPY with the micelle is promoted by the amphiphilic alpha-helical segment of residues Tyr21-Thr32. NPY is located at the lipid-water interface with its C-terminal helix parallel to the membrane surface and penetrates the hydrophobic interior only via insertions of a few long aliphatic or aromatic side-chains. From these data we can demonstrate that the dimer interface of neuropeptide Y is similar to the interface of the monomer binding to DPC-micelles. We speculate that binding of the NPY monomer to the membrane is an essential key step preceeding receptor binding, thereby pre-orientating the C-terminal tetrapeptide and possibly inducing the bio-active conformation.     

Rat brain and kidney metalloendopeptidase: Enkephalin heptapeptide conversion to form a cardioactive neuropeptide,Phe-Met-Arg-Phe-Amide

Rat brain or kidney metalloendopeptidase purified from particulates cleaved Met-enkephalin-Arg⁶-Phe⁷ and its amide at the Gly³-Phe⁴ bond to release Phe-Met-Arg-Phe or the tetrapeptide amide. The latter, a neuropeptide with cardioactive properties, was relatively stable upon further incubation. The metallo-nature of the enzyme was established by inhibition with chelating agents (EDTA, o-phenanthroline) and its endopeptidase nature by cleavage at the Gly³-Phe⁴ bond of pentapeptide enkephalins or precursors such as the heptapeptide, or analogs bearing N- or C-terminal protective groups. Presence of C-terminal amides decreased the rate of hydrolysis. Thiorphan, (DL-3-mercapto-2-benzylpropanoyl)-glycine, competitively inhibited cleavage at the Gly³-Phe⁴ bond of enkephalin (Ki 10 nM). The thiorphan sensitive metalloendopeptidase provides a pathway for conversion of an enkephalin precursor to form a non-opioid peptide of biological interest.     

The neuropeptide Y monomer in solution is not folded in the pancreatic-polypeptide fold

Fluorescence-labelled analogs of NPY, a 36-amino acid peptide amide, were synthesized by solid-phase peptide synthesis and used for fluorescence-resonance energy transfer studies to investigate the conformation. Energy-transfer efficiency measurements in different media at the concentration of 10 microM are in agreement with a model of the NPY structure proposed by NMR studies (performed at millimolar concentration) in which the C-terminal part of the molecule adopts an alpha-helical conformation while the N-terminal part is flexible. According to this model, the alpha-helix is stabilized by intermolecular hydrophobic interactions because of the formation of dimers. The decrease of the peptide concentration causes a shift of the dimerization equilibrium toward the monomeric form. Energy-transfer efficiency measurements performed at lower concentrations do not support the hypothesis of the folding back of the N-terminal tail onto the C-terminal alpha-helix to yield the so-called "PP-fold" conformation. This structure is observed in the crystal structure of avian pancreatic polypeptide, a member of the NPY peptide hormone family, and it has been considered to be the bioactive one. Our results complete the structural characterization of NPY in solution at concentration ranges in which NMR experiments are not feasible. Furthermore, these results open the way to study the conformation of the receptor-bound ligand.     

C-peptide binding to human cell membranes: importance of Glu27

In addition to its established role in proinsulin folding, C-peptide has a function in regulation of cellular activity. The 31-residue peptide influences renal, vascular, and metabolic functions in patients with insulin-dependent diabetes mellitus. Binding to cells has been demonstrated for C-peptide, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirements on the pentapeptide part for C-peptide binding. All pentapeptide residues, E(27)GSLQ(31), were individually replaced with Ala and the capacity of the resulting peptides to displace rhodamine-labelled full-length human C-peptide from human renal tubular cell membranes was determined. This showed that Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal residues had intermediate effects. Morevover, free Glu displaces full-length C-peptide to about 50%, while free Ala, C-peptide(1-26), and the truncated pentapeptide, corresponding to the tetrapeptide G(28)SLG(31), have no displacing capacity. The peptides EVARQ (corresponding to the rat C-terminal pentapeptide) and ELGGGPGAG (corresponding to positions 11-19 of human C-peptide) do not displace human C-peptide. These results indicate that Glu27 of C-peptide is critically involved in binding to cellular targets.     

Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines

Monoiodinated radioligands of the homologous 36-amino acid peptides, neuropeptide Y (NPY) and peptide YY, were prepared by reverse phase high performance liquid chromatography with isocratic elution. [125I-Tyr1]- and [125I-Tyr36]monoiodoNPY bound equally well to a single class of high affinity binding sites on synaptosomal membranes prepared from porcine hippocampus (Kd = 1.0 X 10(-10) M) whereas iodine substitution in Tyr27, for example, partly interfered with the receptor binding. The receptors on the hippocampal membranes did not distinguish between neuropeptide Y and peptide YY either in their monoiodinated or in their unlabeled forms. Six out of twelve human neuroblastoma cell lines had high affinity binding sites for monoiodinated NPY ranging from 2 to 145 X 10(3) sites per cell. The NPY binding to three of the cell lines, SMS-MSN, SMS-KAN, and CHP-234 was of relatively high affinity (Kd = 1.3 to 6.1 X 10(-10) M), and, as in the hippocampal membranes, the long C-terminal fragment, NPY(13-36)peptide was also a relatively potent ligand for these receptors. Two other neuroblastoma cell lines, MC-IXC and CHP-212, expressed NPY receptors characterized by a lower affinity (Kd = 4.8 and 24.6 X 10(-9) M) and negligible cross-reactivity with the C-terminal fragment. It is concluded that monoiodinated radioligands of the tyrosine-rich neuropeptide Y can be prepared and that receptors for these ligands in two apparently different subtypes are found on a series of human neuroblastoma cell lines.     

Y1 and Y2 receptors for neuropeptide Y

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C-terminal fragment, NPY13-36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC-IXC, and a rat pheochromocytoma cell line, PC-12, binds NPY with a dissociation constant (Kd) of a few nanomolar but does not bind NPY13-36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS-MSN, is of higher affinity and binds both NPY and NPY13-36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY-binding sites occur in two subtypes which may represent two types of physiological receptors.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.     

Effects of neuropeptide Y (NPY) at the sympathetic neuroeffector junction. Can pre- and postjunctional receptors be distinguished?

Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effects of a series of NPY-related peptides, namely NPY, PYY, desamido-NPY, and five C-terminal fragments (NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36). NPY and PYY were active in all three assay systems. The C-terminal amide appears to be crucial for maintaining the biological activity, since desamido-NPY was inactive in the three test systems. Interestingly, PYY 13-36 was almost as active as NPY and PYY in suppressing the electrically evoked contractions of the vas deferens; PYY 13-36 was inactive in the two other test systems. None of the shorter fragments had any biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)