Peptides (v.24, #12)

In this study, we analyzed the amino acid pairs affected by mutations in two spike proteins from human coronavirus strains 229E and OC43 by means of random analysis in order to gain some insight into the possible mutations in the spike protein from SARS-CoV. The results demonstrate that the randomly unpredictable amino acid pairs are more sensitive to the mutations. The larger is the difference between actual and predicted frequencies, the higher is the chance of mutation occurring. The effect induced by mutations is to reduce the difference between actual and predicted frequencies. The amino acid pairs whose actual frequencies are larger than their predicted frequencies are more likely to be targeted by mutations, whereas the amino acid pairs whose actual frequencies are smaller than their predicted frequencies are more likely to be formed after mutations. These findings are identical to our several recent studies, i.e. the mutations represent a process of degeneration inducing human diseases.
Keywords: Amino acid pairs; Coronavirus; Mutations; SARS;

We report the preparation of novel building units for backbone cyclization that have the general formula Fmoc-Nα[CH(R)CO2Al]Gly-OH. These building units were prepared by the reductive alkylation method using allyl esters of several amino acids as starting material and hence, respectively, contain the side chain of these amino acids. These N-alkylated Gly building units were incorporated in model backbone cyclic peptides. The resulting crude backbone cyclic peptides were obtained in high degree of purity according to HPLC and mass spectrometric analyses.
Keywords: Backbone cyclization; Difficult coupling; Diketopiperazine; N-alkylated glycine; Reductive alkylation;

Influence of peptide conformation on oligosaccharide binding characteristics—a study using apamin-based chimeric peptide by Cheng Wei Wu; Gurunathan Jayaraman; Kun Yi Chien; Yaw Jen Liu; Ping Chiang Lyu (1853-1861).
Interactions between proteins and heparin play a crucial role in most of the cellular process. Unraveling the forces that govern the formation of these complexes is vital for understanding the specificities involved in these biomolecular events. In the present study, a detailed analysis has been undertaken to evaluate the effect(s) of peptide conformation on heparin-binding, using a chimeric peptide, apaK6—a chimera of a highly stable neurotoxic peptide from honey-bee venom and a de novo designed lysine-rich peptide. The dissociation constants of these peptide–heparin complexes were found to be in the submicromolar range. Comparison of the results obtained from the titration of the disulfide-reduced and disulfide-intact chimeric peptide with various sulfated oligosaccharides, derived from heparin, suggest that the initial structure of the peptide has pronounced effect on the binding affinity, binding modes and also on binding preferences. The results of this study indicate that the heparin-binding specificity of an isolated peptide and that exhibited by the same peptide when present in a globular protein could be significantly different, especially if the isolated peptide undergoes conformational change(s) upon binding to the sulfated oligosaccharides. In addition, such dependency of the binding specificity on the preformed structures could be utilized for the design of high-affinity and sequence-specific heparin-binding polypeptides.
Keywords: Binding specificity; Chimeric peptide; Heparin; Peptide conformation; Peptide design;

Correlations of amino acids in proteins by Qishi Du; Dongqing Wei; Kuo-Chen Chou (1863-1869).
A correlation analysis among 20 amino acids is performed for four protein structural classes (α, β, α/β, and α+β) in a total of 204 proteins. The correlation relationships among amino acids can be classified into the following four types: (1) strong positive correlation, (2) strong negative correlation, (3) weak correlation, and (4) no correlation. The correlation relationships are different for different proteins and are correlated with the features of their structural classes. The amino acids with the weak correlation relationship can be treated as the independent basis functions for the space where proteins are defined. The amino acids with large correlation coefficients are linear correlative with each other and they are not independent. The strong correlation among amino acids reflects their mutual constrained relationship, as exhibited by their relevant structural features. The information obtained through the correlation analysis is used for predicting protein structural classes and a better prediction quality is obtained than that by the simple geometry distance methods without taking into account the correlation effects.
Keywords: Sequential correlation of amino acids; Protein structural Classes; Statistical analysis; Bioinfomatics; Proteomics;

From sinus glands of the Australian crayfish Cherax destructor, two genetic variants of the crustacean hyperglycemic hormone (CHH) were isolated by HPLC and fully characterized by mass spectrometry and Edman sequencing. Both CHH A (8350.38 Da) and CHH B (8370.34 Da) consist of 72 amino acid residues, with pyroGlu as N-terminus and an amidated (Val-NH2) C-terminus. They differ in 14 residues (81% identity). Both sequences are significantly different from those of the hitherto known three CHHs of Astacoidea species (Northern hemisphere crayfish), which among themselves are extremely conserved. This may reflect the long, separate evolution of the Astacoidea lineage and the Parastacoidea (Southern hemisphere crayfish) lineage, to which Cherax belongs. CHH A and CHH B genes are expressed at comparable levels, as indicated by the similar amounts of mature peptides in the sinus gland. In addition to each of the major peptides, which share the identical N-terminal tripeptide pyroGlu-Val-l-Phe, one chiral isoform containing pyroGlu-Val-d-Phe was identified. Compared to the main peptides, the amounts of the d-isoforms are lower, but significant, amounting to 30–40% of l-isoforms. These results demonstrate that two genes can give rise to a total of four different peptides in the secretory terminals of the sinus gland. All peptides gave a highly significant hyperglycemic in vivo response in C. destructor.
Keywords: Crustacean hyperglycemic hormone; Isoforms; Posttranslational modification; d-Amino acid; Mass spectrometry;

Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands by Sam R.J Hoare; Susan K Sullivan; Anil Pahuja; Nicholas Ling; Paul D Crowe; Dimitri E Grigoriadis (1881-1897).
Previous corticotropin releasing factor 1 (CRF1) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF1 receptor heterologously expressed in Ltk cells and endogenously expressed in rat cerebellum. In L-CRF1 cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPγS) bound by [ 125 I ]sauvagine (43 pM, RG); a high affinity state insensitive to GTPγS bound by [ 125 I ]sauvagine (1.4 nM, termed RO); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [ 125 I ]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:RO:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270–330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF1 receptor conformational states in various receptor preparations.
Keywords: Corticotrophin releasing factor; Ligand binding; G-protein-coupled receptor; Urocortin; Guanine nucleotide; Sauvagine; Agonist;

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity by Christine G. Joseph; Andrzej Wilczynski; Jerry R. Holder; Zhimin Xiang; Rayna M. Bauzo; Joseph W. Scott; Carrie Haskell-Luevano (1899-1908).
Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. α-Melanocyte stimulating hormone (α-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.
Keywords: Melanocortin; Melanotropin; Obesity; Agouti; Agouti-related protein; AGRP;

Refractory hypothalamic α-MSH satiety and AGRP feeding systems in rats bearing MCA sarcomas by William T. Chance; Sulaiman Sheriff; Rameshewar Dayal; Ambikaipakan Balasubramaniam (1909-1919).
In pre-anorectic tumor-bearing (TB: methylcholanthrene-induced sarcoma) rats, injection of α-melanocyte stimulating hormone (α-MSH) into the perifornical hypothalamus (PFH) had no significant effect on food intake at a dose (5 μg) that reduced feeding in non-TB control rats. Following the development of anorexia, injection of α-MSH MC3/MC4 receptor antagonists, SHU9119 (1 μg) or 4 μg agouti-related protein (AGRP), stimulated feeding in non-TB rats, while having no significant effect in TB rats. Concentrations of α-MSH were not altered significantly in ventromedial, dorsomedial or lateral hypothalamic areas of TB rats, and proopiomelanocortin (POMC) messenger RNA was not changed in TB rats in these hypothalamic areas. Determination of cytokines by ELISA in non-operated TB and non-TB rats revealed elevated IL-2 in plasma and hypothalamus as well as increased TNF-α in the hypothalamus of anorectic TB rats. IL-1B was not detectable in plasma and was not altered significantly in hypothalamus of TB rats. These results suggest that the POMC α-MSH satiety system is refractory in TB rats, even prior to the onset of anorexia. This change in MC3/MC4 receptor response does not appear to be secondary to alterations of endogenous α-MSH in TB rats. Cytokine involvement in the altered response to MC3/MC4 receptor stimulation and blockade is a possibility, since TNF-α and IL-2 were increased in hypothalamus of anorectic TB rats. Therefore, these results suggest major alterations in POMC neuropeptide systems in TB rats as anorexia progresses. Although these changes do not appear to have occurred due to grossly-altered concentrations of α-MSH, elevated cytokine activity in the hypothalamus may be an important factor. Due to the complex multi-factorial nature of feeding control, additional factors are likely to be involved in cancer anorexia.
Keywords: Anorexia; Cachexia; α-MSH; Agouti; Cytokines; POMC;

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus by Jian-Lian Guan; Qing-Ping Wang; Haruaki Kageyama; Fumiko Takenoya; Tohru Kita; Takashi Matsuoka; Hisayuki Funahashi; Seiji Shioda (1921-1928).
Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.
Keywords: Arcuate nucleus; Electron microscopy; Immunocytochemistry; Synapse; Ghrelin; Neuropeptide Y.;

Adaptation to low-protein diet increases inhibition of gastric emptying by CCK by Véronique Leray; Jean-Pierre Segain; Christine Cherbut; Jean-Paul Galmiche (1929-1934).
Chronic nutritional disorders such as protein malnutrition are associated with delayed gastric emptying and increased postprandial cholecystokinin (CCK) levels. This study investigated the mechanisms involved in gastric emptying adaptation to low-protein diet. Two groups of 12 rats were adapted to a low-protein (LPD) or standard diet (SD) for 3 weeks. As compared to rats fed a SD, in rats adapted to a LPD gastric emptying was delayed, whereas postprandial CCK levels were increased. LPD enhanced antral muscle contractile response to CCK and cerulein without altering response to acetylcholine. This increased contractility was associated with up-regulation of CCK-A receptor mRNA levels in antral muscle. Our data suggest that modulation of gastric emptying after adaptation to a low-protein diet involves up-regulation of both CCK-A receptors and CCK-induced contraction of antral smooth muscle.
Keywords: Gastric emptying; Malnutrition; Cholecystokinin receptors; Gastric muscle contractility;

Effects of chronic ethanol on brain and serum level of methionine enkephalin by William A. Banks; Kathleen M. Wolf; Michael L. Niehoff (1935-1940).
Most evidence agrees that levels of methionine enkephalin (Met-Enk) in brain are inversely correlated with ethanol drinking and withdrawal seizures. One area of discrepancy is the effect of chronic ethanol administration on the level of immunoactive Met-Enk in brain, with some authors reporting increased and others reporting decreased levels. These reports differed greatly in terms of method of ethanol administration, species used, length of time ethanol was administered, and the region of brain examined. We found that all studies could be resolved by considering only length of time ethanol was administered, with Met-Enk levels first increasing and then decreasing. We tested this finding by determining the effect of 4–56 days of ethanol delivered in liquid feed on levels of brain Met-Enk. We found that brain levels of Met-Enk peaked after 7 days of ethanol ingestion and declined to levels lower than control by 28 days. Exposure to ethanol abolished a correlation between brain and serum levels of Met-Enk which occurred in controls. HPLC showed that whereas 100% of immunoactivity eluted in the position of Met-Enk in controls, only about 50% eluted as Met-Enk in mice exposed to ethanol. These results support the hypothesis that exposure to ethanol alters brain Met-Enk in a way consistent with the reinforcement of physical dependence.
Keywords: Blood-brain barrier; Alcoholism; Peptide; Addiction; CNS;

β-Endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid β-endorphin receptors by Elena V Navolotskaya; Yulia A Kovalitskaya; Yury A Zolotarev; Nina Yu Kudryashova; Elena N Goncharenko; Alexander A Kolobov; Elena A Kampe-Nemm; Natalia V Malkova; Vladimir V Yurovsky; Valery M Lipkin (1941-1946).
β-Endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (K d=31.6±0.2 nM, B max=37.4±2.2 pmol/mg protein). Immunorphin at concentrations of 10−9 to 10−6  M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10–100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.
Keywords: β-Endorphin; Nonopioid β-endorphin receptor; Adrenal cortex;

Functional properties of PFR(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors by Mia Engström; Siegfried Wurster; Juha-Matti Savola; Pertti Panula (1947-1954).
The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [ 35 S ]guanosine-5′-O-(3-thio)triphosphate ([ 35 S ]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [ 35 S ]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.
Keywords: Neuropeptide FF; Antagonist; PFR(Tic)amide; BIBP3226; Neuropeptides; [ 35 S ]GTPγS binding assay; NPFF2 receptor; Super-agonist;

β-Lactotensin and neurotensin rapidly reduce serum cholesterol via NT2 receptor by Rena Yamauchi; Kousaku Ohinata; Masaaki Yoshikawa (1955-1961).
β-Lactotensin, a neurotensin NT2 agonist derived from β-lactoglobulin, has hypocholesterolemic activity after administration for 2 days at a dose of 30 mg/kg (i.p.) or 100 mg/kg (p.o.) for 2 days in mice fed a high-cholesterol/cholic acid diet. The onset of hypocholesterolemic activity of β-lactotensin was observed 90 min after a single i.p. or p.o. administration at the same dose as described above. Neurotensin also induced hypocholesterolemic activity 90 min after single i.p. administration at a dose of 2 μg per mouse but was ineffective after oral administration. The rapid onset of hypocholesterolemic activities of β-lactotensin and neurotensin was blocked by levocabastine (50 μg/kg), an NT2 antagonist, and raclopride (0.5 mg/kg), a dopamine D2 antagonist.
Keywords: Neurotensin; β-Lactotensin; β-Lactoglobulin; Cholesterol; NT2-receptor; Levocabastine; Dopamine; Raclopride;

The cardiac effects of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) as well as the possible signaling pathways were investigated. In the isolated perfused rat heart, infusion of AM (10−11 to 10−8  M) and PAMP (10−11 to 10−8  M) for 10 min, alone or in combination, induced concentration-dependent decreases in the left ventricular pressure (LVP), LVP±dp/dt max of the hearts. The effects were attenuated by N ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide (NO) synthase. ADM and PAMP alone or in combinations increased the coronary fluid (CF), which could be antagonized by l-NAME. Pretreatment of H89, an inhibitor of protein kinase A (PKA), failed to alter the AM- or PAMP-induced decreases in LVP and LVP±dp/dt max, but further promoted the AM or PAMP increased CF. The cAMP content in left cardiac ventricle was increased significantly by ADM infusions but not by PAMP. There was no statistical difference in cAMP contents with ADM administrated alone from those combined with ADM and PAMP. In conclusion, this study reveals that ADM and PAMP infused alone or in combinations inhibited the function of rat hearts in vitro, which may be partly involved with the NOS/NO pathway, rather than cAMP/PKA.
Keywords: Adrenomedullin; Proadrenomedullin N-terminal 20 peptide; N ω-nitro-l-arginine methyl ester; Isolated rat heart;

Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter by M.P. Majowicz; L.V. Gonzalez Bosc; M.F. Albertoni Borghese; M.F. Delgado; M.C. Ortiz; N. Sterin Speziale; N.A. Vidal (1971-1976).
Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491–5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069–75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na+ and d-glucose. Using α- 14 C -methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant (K 0.5∼2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.
Keywords: SGLT2; ANP; ET-3; cGMP; Brush border membrane vesicles; Kidney;

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme by Pika Meško Brguljan; Vito Turk; Nina Cimerman; Jože Brzin; Igor Križaj; Tatjana Popovič (1977-1984).
Highly purified human brain cathepsin H (EC was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1–6), dynorphin (1–13), α-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid d-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1–13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1–6), α-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [Δ1–10] variant, already known to be co-localized in astrocytes, since the K i values for the inhibition are in the 10−10  M range.
Keywords: Cathepsin H; Cystatin C; Truncated cystatin C; Neuropeptide; Bradykinin; Brain; Human;

The role of calcium channels in substance P-induced contractile response in the rat iris by Astor Grumann; Ricardo V. Alves; João B. Calixto (1985-1991).
This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca2+, by a mechanism which might involve the T-type calcium channels.
Keywords: Rat iris; Substance P; Calcium channels; Calcium antagonists;